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obálka 2-3str 10.6.2003 18:09 Stránka 2
01 Úvod 10.6.2003 11:39 Stránka 1
TH
13
INTERNATIONAL CONFERENCE
ON
CYTOCHROMES P450
Biochemistry, Biophysics and Drug Metabolism
Supported by the
European Commission, High-Level Scientific Conferences,
HPCF-CT-2002-00150
and by the
International Union of Biochemistry and Molecular Biology
(support No. IG215)
PROGRAM / BOOK OF ABSTRACTS
June 29 - July 3, 2003
www.cyp2003.cz
01 Úvod 10.6.2003 11:39 Stránka 2
Organized by
Czech Chemical Society
Co-organizers
Palack˘ University, Olomouc
Czech Society for Experimental
and Clinical Pharmacology and Toxicology
Under the auspices of
Ministry of Education, Youths and Sports
of Czech Republic
Ministry of Industry and Trade, Czech Republic
CONTENTS
Welcome to Prague
page
3
Committees
page
4
Important Contacts
page
4
Sponsors/Exhibitors
page
5
Scientific Program
page
6 – 11
Poster Schedule
page
12 – 24
General Information
page
25 – 26
Social Program
page
26 – 27
Tourist Program
page
27 – 28
Accommodation
page
28
Abstracts
page
29 – 209
Author Index
page
210 – 215
List of Participants
page
216 – 231
2
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WELCOME IN PRAGUE
Dear Colleagues:
We have a great pleasure to welcome you as participants of the 13th International
Conference on Cytochromes P450 in Prague. The P450 community meets in this city for
the second time - after fifteen years. In 1988, Prague hosted the International Congress
of Biochemistry and Molecular Biology, where symposia dedicated to cytochromes P450
attracted many of you.
Since that time, many things have changed and happened. Our beloved P450 however
remains to be one of the most interesting, intriguing and beautiful topics of
contemporary biochemistry, biophysics and biochemical pharmacology. We see the
rapid development of molecular biology, genomics and proteomics also in the field of
P450. There is a growing interest in the medical community in the practical applications
of P450 science - for example, the drug interactions based on P450-mediated
metabolism can elucidate many unwanted drug effects. Traditionally, the core of our
Conferences remains the same - fundamental questions of P450 biochemistry and
biophysics.
We sincerely hope that you will find the scientific program interesting and that you will
enjoy also the social events, accompanying the Conference. Prague is now an
attractive city offering also a wide variety of pleasant trips and tourist attractions.
On behalf of the Organizing Committee,
Jifií Hudeãek
Secretary of the Organizing Committee
Pavel Anzenbacher
Chairman of the Organizing Committee
INVITATION TO DALLAS
The next, 14th International Conference on Cytochromes P450, will be held in
Dallas, Texas, USA, May 31 - June 4, 2005.
For more information, please visit www.p450dallas2005.us
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COMMITTEES
INTERNATIONAL SCIENTIFIC COMMITTEE
F. Peter Guengerich (USA) - Chairman
Pavel Anzenbacher (CZ), Alexander Archakov (R), Rita Bernhardt (D),
Minor J. Coon (USA), John Dawson (USA), Ronald Estabrook (USA),
Yoshiaki Fujii-Kuriyama (J), Frank Gonzalez (USA), Reinhard Lange (F),
Bettie S.S. Masters (USA), Patrick Maurel (F), Olavi Pelkonen (FIN),
Steve Sligar (USA), Wolf-Dieter Woggon (CH)
ORGANIZING COMMITTEE
Pavel Anzenbacher - Chairman (Palack˘ University, Olomouc)
Jifií Hudeãek - Secretary (Charles University, Prague)
Petr Hodek (Charles University, Prague), Jifiina Martínková (Charles
University, Hradec Králové), Milan Miko (Slovak Technical University,
Bratislava), Pavel Souãek (National Institute of Public Health, Prague),
Marie Stiborová (Charles University, Prague), Monika ·enderová
(Congress Business Travel, Prague), Vilím ·imánek (President,
Czech Chemical Society)
NATIONAL ADVISORY COMMITTEE
Pavel Anzenbacher - Chairman (Palack˘ University, Olomouc)
Hassan Mohammad Farghali (Charles University, Prague),
Eva Hada‰ová (Masaryk University, Brno), Miloslav Kr‰iak (Charles University,
Prague), Eva Kvasniãková (Charles University, Hradec Králové),
Jaroslav Kvûtina (Institute of Experimental Biopharmaceutics,
Hradec Králové), Miroslav Machala (Research Institute of Veterinary
Medicine, Brno), Franti‰ek Perlík (Charles University, Prague, President
of the Czech Society for Experimental and Clinical Pharmacology and
Toxicology), Vilím ·imánek (Palack˘ University, Olomouc, President of the
Czech Chemical Society), Tomá‰ Zima (Charles University, Prague)
IMPORTANT CONTACTS
CONFERENCE SECRETARIAT
„CYTOCHROMES P450“
Congress Business Travel Ltd. (CBT)
Lidická 43/66
150 00 Prague 5, Czech Republic
Phone: +420-224 942 575, 224 942 579
Fax: +420-224 942 550
E-mail: [email protected]
CHAIRMAN OF THE ORGANIZING
COMMITTEE
Dr. Pavel Anzenbacher
Department of Pharmacology
Faculty of Medicine, Palack˘ University
Hnûvotínská 3
775 15 Olomouc, Czech Republic
E-mail: [email protected]
4
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SPONSORS & EXHIBITORS
The Organizing Committee is pleased to acknowledge the
generous support of the following sponsors and exhibitors:
●
ABBOTT Laboratories, s. r. o.
●
AVENTIS PHARMACEUTICALS, Inc.
●
BIO-RAD, s. r. o.
●
BIOTECH, a.s.
●
CZECH AIRLINES - SKY TEAM
●
DESITIN Pharma, s. r. o.
●
ELI LILLY and Co.
●
EUROPEAN COMMISSION,
Research Directorates,
Human Potential Program
●
IN VITRO TECHNOLOGIES, Inc.
●
INTERNATIONAL SOCIETY FOR STUDY
OF XENOBIOTICS
●
INTERNATIONAL UNION
OF BIOCHEMISTRY AND MOLECULAR
BIOLOGY
●
JANSSEN-CILAG, JOHNSON & JOHNSON, s.r.o.
●
JOHNSON & JOHNSON
PHARMACEUTICAL
RESEARCH & DEVELOPMENT,
a division of Janssen Pharmaceutica n.v.
●
MERCK & Co., Inc.
●
P-LAB, s.r.o.
●
PFIZER, Inc.
●
SCHERING-PLOUGH
RESEARCH INSTITUTE
●
SCHOELLER PHARMA, s.r.o.
●
SIGMA-ALDRICH, s.r.o.
●
STABILIGEN S.A.
●
QUINTILES Ltd.
●
WYETH WHITEHALL CZECH, s.r.o.
5
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SCIENTIFIC PROGRAM
LANGUAGE
The official Conference language will be English without
simultaneous translation.
ORAL COMMUNICATIONS
Oral communications will consist of Invited Lectures, Forum of
Young Scientists and Round Table Discussion on topics which
deserve to be discussed in more detail. Overhead and data
projection (IBM PC compatible, MS PowerPoint) will be available.
The speakers are kindly requested to consult their presentations
in the Preview Room at least 30 minutes before their presentation is
scheduled, but preferably before the start of the respective session.
Round Table discussion is supposed to attract participants wishing
to discuss their (innovative) opinion and views. The organizers
welcome the proposals for a 10 min. talks. Please contact the
Conference Secretary Dr. Jifií Hudeãek.
PROGRAM OF THE CONFERENCE
M o n d a y, J u n e 3 0
8:00 - 8:15
Conference Opening
Hall A:
Plenary lectures: Structural Aspects (Chair: P. R. Ortiz de Montellano, J. R. Halpert)
8:15 - 8:45
8:45 - 9:15
JR Halpert
E Scott
9:15 - 9:45
J Cosme
9:45 - 10:15
EF Johnson
10:15 - 10:30
Coffee break
10:30 - 11:00
11:00 - 11:25
PR Ortiz de
Montellano
TL Poulos
11:25 - 11:50
LL Wong
11:50 - 12:15
I Schlichting
12:15 - 14:00
LUNCH, POSTERS
ML01 Structural basis of P450 2B specificity
ML02 A1.6 Å crystal structure of P450 2B4: novel features and
implications
ML03 Insights on the topology of the active site of human
cytochrome 2C9 determined by X-ray crystallography
ML04 Structural studies of substrate binding to CYP2C drug
metabolising P450s
ML05 Radicals and stressed bacterial P450 systems
ML06 Structures of P450epoK, putidaredoxin, and putidaredoxin
reductase
ML07 Crystal structure - based engineering of substrate
recognition in P450cam catalysis
ML08 Structural aspects of ligand binding to procaryotic and
fungal P450
6
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Hall A:
Nitric Oxide Synthase (Chair: B. S. S. Masters, D. Mansuy)
14:00 - 14:30
BS Masters
14:30 - 15:00
15:00 - 15:30
D Mansuy
R Lange
15:30 - 15:45
Coffee break
15:45 - 16:15
16:15 - 16:45
I Sagami
CS Raman
Hall B:
ML09 Revelations of nitric oxide synthase modulation:
Hidden messages in sequence and structure
ML10 Formation of NO from xenobiotics
ML11 Tracking reactive intermediate states of NO synthase
under extreme conditions of temperature and pressure
ML12 Regulation of electron transfer in neuronal NO synthase
ML13 Structural and functional evidence for the evaluation of
prokaryotic NO synthases from an ancestral cytochrome
P450
Bioinformatics (Chair: A. Archakov, J. A. Peterson)
14:00 - 14:30
A Archakov
14:30 - 15:00
JA Peterson
15:00 - 15:30
AS Ivanov
15:30 - 15:45
Coffee break
15:45 - 16:15
R Wade
16:15 - 16:45
16:45 - 17:15
S Kelly
AV Lisitsa
20:00
CONCERT
ML14 Bioinformatic insight into unity and diversity of
cytochromes P450
ML15 From sequence to structure and then function: How much
of a leap is warranted?
ML16 General trends in 3D modeling of cytochromes P450
ML17 Cytochrome P450 friends and foes: Complex relationships under computational analysis
ML18 New insights into microbial CYP biodiversity
ML19 Balance sheet for cytochrome P450 knowledgebase:
2000 protein sequences, 10 000 chemical substances
Tu e s d a y, J u l y 1
Hall A:
Plenary lectures: Enzymology and Mechanisms 1 (Chair: F. P. Guengerich, J. H. Dawson)
8:30 - 9:00
9:00 - 9:30
FP Guengerich
S Shaik
9:30 - 10:00
SG Sligar
10:00 - 10:15
Coffee break
TL01 Rate-limiting steps in cytochrome P450 reactions
TL02 The protein, the active species and the chameleon:
Reactivity patterns of oxygen transfer by cytochrome P450
TL03 P450’s peccant proton pathways: Periclesian control of
active oxygen
7
01 Úvod 10.6.2003 11:40 Stránka 8
Hall A:
Enzymology and Mechanisms 2 (Chair: F. P. Guengerich, J. H. Dawson)
10:15 - 10:45
10:45 - 11:15
JH Dawson
I Morishima
11:15 - 11:45
11:45 - 12:15
WD Woggon
A Chougnet
Regulation of P450 Expression (Chair: P. Maurel, Y. Fujii-Kuriyama)
Hall B:
10:15 - 10:45
10:45 - 11:15
11:15 - 11:45
11:45 - 12:15
TL04 Mechanistic studies of cytochromes P450
TL05 Molecular mechanism of P450cam catalyzed oxygenation
reaction regulated by the association with putidaredoxin
TL06 Design and synthesis of a new cytochrome P450 mimics
TL07 Design and synthesis of new fluorescent probes for
evaluation of CYP3A4 drug-drug interactions
P Maurel
TL08 Overview of CYP gene regulation within a tangle of
regulatory networks
Y Fujii-Kuriyama TL09 Molecular mechanisms of transactivations of Ah
receptor in target gene expression
JM Pascussi
TL10 Physiopathological factors affecting PXR and CAR gene
expression
A Anderson
TL11 Induction of rat CYP2B genes by phenobarbital-like
inducers: Something old and something new
12:15 - 14:15
LUNCH, POSTERS
16:00
STEAMBOAT PARTY
We d n e s d a y, J u l y 2
Hall A:
Plenary lectures: P450 System: Component Interactions 1 (Chair: R. Bernhardt, P. M. Champion)
8:15 - 8:45
R Bernhardt
8:45 - 9:15
PM Champion
9:15 - 9:45
WL Backes
9:45 - 10:00
Coffee break
WL01 Mitochondrial cytochrome P450 systems: New insights
into structure and function
WL02 Resonance Raman and femtosecond coherence
spectroscopy of P450: Interactions with reductase,
oxygen and nitric oxide
WL03 Interactions among reductase and multiple P450
enzymes in mixed reconstituted systems
Applications 1 (Chair: P. Anzenbacher, A.Guillouzo)
Hall A:
10:00 - 10:30
10:30 - 11:00
P Anzenbacher
U Fuhr
11:00 - 11:30
O. Pelkonen
11.30 - 12:00
DJ Waxman
WL04 Experimental in vitro models of drug metabolism
WL05 Should we personalize the drug therapy according to
cytochrome P450 genotype and/or phenotype?
WL06 In vitro to In vivo correlations - a critical assessment of
various approaches to measure drug metabolism and
metabolic interactions
WL07 P450, anti-cancer drugs and gene therapies
8
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Hall B:
P450: In Situ Function 1 (Chair: M. Waterman, R. Tyndale)
10:00 - 10:30
10:30 - 11:00
MR Waterman
D Rozman
WL08 CYP51: Structure, function, evolution
WL09 Lanosterol 14alpha- demethylase CYP51, cholesterol
and reproduction
WL10 P450 humanized mice: Novel tools for the study of drug
and carcinogen metabolism and the search for
endogenous human P450 substrates and functions
WL11 Mechanism of the formation and maintenance of the
functional zonation in the adrenal cortex
11:00 - 11:30
FJ Gonzalez
11:30 - 12:00
F Mitani
12:00 - 14:00
LUNCH, POSTERS
Applications 2 (Chair: P. Anzenbacher, A. Guillouzo)
Hall A:
14:00 - 14:30
M Stiborová
14:30 - 15:00
A Guillouzo
15:00 - 15:30
F Oesch
15:30 - 15:45
Coffee break
15:45 - 16:15
S Coecke
16:15 - 16:45
CJ Henderson
WL11 Utilization of cytochromes P450 to explain of a new
mode of action of anti-cancer drug ellipticine
WL12 Dual effects of the chemoprotective agent oltipraz on
cytochromes P450
WL13 Trans-species regenerating or stem cell derived
hepatocyte-like cells: A perspective to solve the
availability problem for human hepatocytes?
WL14 Metabolism: a bottle-neck for in vitro regulatory toxicity
tests and testing strategies
WL15 Applications of transgenic models in drug metabolism
P450: In Situ Function 2 (Chair: M. Waterman, R. Tyndale)
Hall B: P450:
14:00 - 14:30
HW Strobel
WL16 Cytochrome P450 4F5: Response and role following
brain trauma
WL17 Commonly used drugs regulate CYP enzymes within the
brain
14:30 - 15:00
RF Tyndale
15:00 - 15:15
Coffee break
15:15 - 15:45
JH Capdevila
15:45 - 16:15
NG Avadhani
19:00
FAREWELL DINNER
WL18 The arachidonic acid monooxygenases: P450eikosanoids as endogenous regulators of organ and body
physiology
WL19 Chimeric signals of xenobiotic-inducible CYPs:
Mechanism of mitochondrial targeting and functional
implications
9
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T h u r s d a y, J u l y 3
Hall A:
P450 system: component interactions 2 (Chair: R. Bernhardt, P. M. Champion)
8:30 - 9:00
DC Lamb
9:00 - 9:30
AW Munro
10:00 - 10:15
Coffee break
P450 Genomics/Proteomics (Chair: M. Ingelman-Sundberg, U. M. Zanger)
Hall B:
8:30 - 9:00
CL02 Regulation and interaction of redox pertners with
actinomycete CYPs
CL03 Mechanisms and regulation of electron transfer in
cytochrome P450 redox systems
9:00 - 9:30
M Ingelman
-Sundberg
UM Zanger
CL04 Mechanisms and consequences of interindividual
variation in cytochrome P450 functional genomics
CL05 Role of genetic polymorphism, drug exposure and sex
for expression and function of hepatic cytochromes P450
9:30 - 10:00
H Ohkawa
CL06 Environmental genomics on plant P450 species
metabolizing herbicides
10:00 - 10:15
Coffee break
10:15- 12.30
FORUM
Hall A:
Round Table Discussion – Moderators: R. Lange, P. Hodek
10:15 - 10:40
S Narashimhulu CL07 Differential behavior of the two subsites of P450 active
site in binding of substrate and product
10:40 - 12:30
Speakers will be announced
Hall A :
Summary, closing
12:45 - 13:15
Summary
13:15 - 13:30
Closing Ceremony
OF
YOUNG SCIENTISTS – Moderators: J Wojcikowski, L. Antonoviç
The Organizing Committee gratefully acknowledges the sponsoring of following lectures:
TL08 (JOHNSON & JOHNSON PHARMACEUTICAL RESEARCH & DEVELOPMENT)
WL05 (ABBOTT Laboratories, s. r. o.),
WL06 (IN VITRO TECHNOLOGIES, Inc.),
ML05 (WYETH WHITEHALL CZECH, s.r.o.)
10
01 Úvod 10.6.2003 11:40 Stránka 11
FORUM OF YOUNG SCIENTISTS
Oral communications of:
W. E. Straub, A. R. Dunn, C. de Graaf, Z. Guan, O. Roitel,
P. Meinhold, T. S. Wong, M. Fink, L. M. Podust, L. B. von
Weymarn, M. Duisken, A. L. Sukhodub, K. J. McLean,
S. Pronko, N. Pons.
In addition to the authors listed above, the following EU Grant
recipients are expected to present a short outline of their
results:
A. Bonifacio, S. Catania, E. O’Donnell, J. Hodis, M. Tesafiová,
D. Herman, M. ·tûrba, O. Georgescu.
POSTERS
POSTER PRESENTATIONS
Each day, space and time will be assigned to poster presentations. Posters are located at the balcony and in the corridors (follow the arrow signs). The authors are kindly requested to be available at their poster presentation for questions and discussion for at least 30 minutes on the announced
day between 12.15 and 14.00. Pins will be provided, poster
size 2 m (height) x 1 m. The authors are requested to set
up their posters in the morning (starting from 8.00 on the
announced day) and remove them after the end of the session
(before 17.00).
11
01 Úvod 10.6.2003 11:40 Stránka 12
D. R. Davydov, A. Bochkareva, S. Kumar, and
J. R. Halpert.
POSTER SCHEDULE
MP09
Molecular modeling of alkoxyresorufin oxidation by
P450 1A1, 1A2 and their mutants
S. S. Ericksen, and G. D. Szklarz
M o n d a y, J u n e 3 0
MP01
Axial ligation for enhancing selectivity in catalytic
epoxidation by cytochrome P450 models
C. K. Chang, T. S. Lai, and L-L Yeung
MP10
Can P450 BM3 turn over substrates typically metabolised by class II human P450s? Evidence on
the functional relationship of P450 BM3 with the
human enzymes
A. Fantuzzi, and G. Gilardi
MP02
Functional analysis of CYP2D6.31 - Arg440His
substitution diminishes enzyme activity by disrupting redox partner interaction
D. Allorge, D. Breant, J. Harlow, J. Chowdry,
J.-M. Lo-Guidice, D. Chevalier, C. Cauffiez,
M. Lhermitte, F. E. Blaney, G. T. Tucker, F. Broly,
and S. W. Ellis
MP11
The role of Phe120 in CYP2D6 substrate binding
J. U. Flanagan, C. Kemp, M. Sutcliffe,
M. J. I. Paine, G. C. K. Roberts, and C. R. Wolf
MP03
Covalent heme binding to CYP4B1 via Glu310 and
a carbocation porphyrin intermediate
B. R. Baer, Y.-M. Zheng, M. J. Cheesman,
N. Moguilevsky, K. L. Kunze, and A. E. Rettie
MP12
Resonance Raman spectra reveal subtle differences in heme structure of P450 enzymes
J. Hudeãek, P. Anzenbacher, T. Shimizu,
A.W. Munro, and T. Kitagawa
MP04
α-Naphthoflavone as a model ligand of the active
site of CYP3A
L. Bofiek-Dohalská, P. Hodek, and M. Stiborová
MP13
Fluorescence correlation spectroscopy study of
interaction between CYP 3A4 and coumarin 6
M. Bene‰, J. Pekárek, M. Hof, P. Anzenbacher,
J. Hudeãek
MP05
Structure-function relationship in cytochrome P450
CYP 2C11
C. Celier, C. Biagini, R. C. Maroun, and R. Philpot
MP14
Probing the alterations in the active site of P420cam:
high pressure studies on the P450cam-ligands and
-inhibitors complexes
G. Hui Bon Hoa
MP06
Camphor hydroxylation by cytochrome P450cam:
A theoretical study by hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) method
S. Cohen, S. Shaik, J. C. Schöneboom, H. Lin,
and W. Thiel
MP15
CYP enzymes: moving towards virtual screening
N. Jourdan, and A. Mancy
MP07
Cytochrome P450 3A4 allosteric mechanism studied by high-pressure spectroscopy
D. R. Davydov, and J. R. Halpert
MP16
Heme pocket compressibility in P450cam-CO: high
pressure FTIR studies on the CO ligand stretch vibration
C. Jung, B. Canny, J. C. Chervin, and G. Hui Bon Hoa
MP08
A charge-pairing bundle around cys-154 is pivotal for
the allosteric mechanism in cytochrome P450eryF
MP17
Intermediate radical formation by the iron-oxo
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01 Úvod 10.6.2003 11:40 Stránka 13
complex produced in the reaction of cytochrome
P450cam with oxidants
C. Jung , V. Schünemann, F. Lendzian, J. Contzen,
A.-L. Barra, and A. X. Trautwein
D. Murtazina, S. Graham, J. A. Peterson,
I. Bjorkhem, and I. Pikuleva
MP26
Metabolism prediction for cytochrome P450
I. Zamora, M. Ridderström, R. Vianello,
G. Cruciani, and T. B. Andersson
MP18
Microliter scale surface enhanced resonance Raman scattering on cytochrome P450 BM3
P. H. J. Keizers, A. Bonifacio, J. N. M. Commandeur, G. van der Zwan, C. Gooijer, S. M. van der
Vies, and N. P. E. Vermeulen
MP27
Alteration of CYP 4A4 regiospecificity by site-directed mutagenesis of residues in the substrate
binding channel
L. J. Roman, M. de la Garza, M. Mock, D. Harris,
and B. S. Masters
MP19
Membrane topology of P45017α studied by chemical modifications and mass-spectrometry
H. Kaneko, S. Izumi, T. Yamazaki, T. Hirata, and
S. Kominami
MP28
Crystal structure of putidaredoxin
I. Sevrioukova, C. Garcia, H. Li, B. Bhaskar, and
T. L. Poulos
MP20
The AFM study of complex formation within the cytochrome P450scc-containing system
V. Kuznetsov, Y. Ivanov, S. Usanov, and A. Archakov
MP29
Thiols prevent P420 formation and aggregation in
25-hydroxyvitamin D3 24-hydroxylase
M. J. Theisen, A. Annalora, S. A. Beretta, A. Pastuszyn, E. D. Matayoshi, J. L. Omdahl, M. L. Chiu
MP21
The role of ferric-peroxo reactivity at the crossroads of P450 catalysis
T. M. Makris, I. G. Denisov, I. Schlichting, and
S. G. Sligar
MP30
The selective inhibition of cytochrome P450 1 enzymes by the derivatives of rutaecarpine
Y.-F. Ueng, M.-J. Don, D. F. V. Lewis, S.-Y. Wang,
and M.-W. Tsai
MP22
CYP2D6: role of Glu-216 and Asp-301 in quinidine
binding
L.A. McLaughlin, M. J. Sutcliffe, M. J. I. Paine,
G. C. K. Roberts, and C. R. Wolf
MP31
The protein stabilization of coumarin 7-hydroxylase
by its own inhibitors
P. Viitala, O. Pelkonen, and R. Juvonen
MP23
Crystallographic studies of perdeuterated P450cam
F. Meilleur, M.-T. Dauvergne, M. Haertlein, I. Schlichting, and D. Myles
MP32
Validation of a 3D-rebuilt P450 3A4 structure with cyclopeptide metabolism: implementation of new techniques
involving soft-restrained dynamics docking simulations
gives new insights on the multiple substrate specificity
F. André, N. Loiseau, M. Delaforge
MP24
Crystal structures of epothilone-D bound, epothilone-B bound, and substrate-free forms of cytochrome P450epoK
S. Nagano, H. Li, H. Shimizu, C. Nishida, H. Ogura,
P. R. Ortiz de Montellano, and T. L. Poulos
MP33
Substrate dynamics in cytochrome P450-BM3 - dynamics of substrate binding by automated docking
and molecular dynamics simulation
K. A. Feenstra, C. de Graaf, J. Starikow,
J. N. M. Commandeur, and N. P. E. Vermeulen
MP25
Identification of the substrate-contact residues in
cytochrome P450 27A1
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01 Úvod 10.6.2003 11:40 Stránka 14
MP34
A QM/MM study of cytochrome P450: the influence
of the enzyme environment on specific steps in the
catalytic cycle
A. R. Groenhof, M. Swart, A. W. Ehlers, and
K. Lammertsma
L. Ferrari, A. Thompson, D. Froenzler, S. Visvikis,
and A. M. Batt
MP43
+
Comparison of NADPH-, NAD - and UDP-glycoside-dependent biotransformation of bohemine
and roscovitine in vitro
K. âervenková, Z. Chmela, M. Belejová,
L. Uherková, M. Rypka, and D. Riegrová
MP35
Aspects on evolution of the CYP51 family
T. ReÏen, N. Debeljak, D. Kordi‰, and D. Rozman
MP44
Biotransformation of olomoucine-type cyclin-dependent kinase inhibitors by precission-cut tissue
slices from different animal species
K. âervenková, Z. Chmela, and M. Rypka
MP36
Identification of novel human cytochrome P450s,
CYP4Z1 and the transcribed pseudogene CYP4Z2P
M. Rieger, R. Ebner, A. Kiessling, J. Rohayem,
M. Schmitz, A. Temme, E. P. Rieber, and
B. Weigle
MP45
Significant role of CYP2A and CYP3A in biotransformation of bohemine by mouse liver microsomes in
vitro
M. Rypka, Z. Chmela, K. âervenková, K. Lemr,
and D. Riegrová
MP37
Characterization of Arabidopsis thaliana CYP711A1
M. Shimoji, F. Durst, I. Benveniste, and
R. Morgenstern
MP46
Carboxylic acid is the main product of roscovitine
biotranformation in mice in vivo. Comparison with
bohemine
Z. Chmela, M. Rypka, K. âervenková, K. Lemr,
D. Riegrová, and J. Vesel˘
MP38
Inducible cytochrome P 450 (CYP2E1) by combined administration of acetaminophen and caffeine
V. Kovalenko, and A. Voronina
MP39
Molecular modeling and site-directed mutageneses defining catalytic sites in insect P450s metabolizing allelochemicals and insecticides
J. Baudry, X. Li, Z. Wen, L. Pan, M. R. Berenbaum,
and M. A. Schuler
MP47
CYP2D is involved in the neurosteroidogenesis in
the brain
W. Kishimoto, T. Hiroi, M. Osada, S. Imaoka,
T. Igarashi, and Y. Funae
MP40
Transcript profiling of Arabidopsis thaliana P450s
S. Ali, H. Duan, Y. Ferhatoglu, M. Band,
D. Werck-Reichhart, and M. A. Schuler
MP48
Analysis of cytochrome P450s in a pyrethroid resistant
strain of Helicoverpa armigera (cotton bollworm)
V. Grubor, and D. G. Heckel
MP41
P450-dependent alkane monooxygenase of Acinetobacter sp. EB104 is encoded in a composite
transposon of the plasmid pAC450
M. Ludewig, C. Schwarz, O. Asperger, S. Pääbo,
and U. Hahn
MP49
Influence of recipient gender on cytochrome P450
isoforms expression and activity in intrasplenic fetal
liver tissue transplants in comparison to liver in rats
A. Lupp, S. Hugenschmidt, and D. Müller
MP42
CYP2C19 do not contribute to blood pressure
C. Bertrand-Thiebault, B. Marie, S. Droech,
MP50
High-throughput LC-MS/MS method for CYP2D6
phenotyping with dextromethorphan
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T. E. Mürdter, G. Schänzle, G. Heinkele, K. Endrizzi,
C. Marx , M. Schwab, M. Eichelbaum, and U. Zanger
MP59
Cocaine, but not metamphetamine causes a concomittant decrease of CYP2C and constitutive androstane receptor (CAR) expressions in human
astrocytoma cell line U373 MG
C. Malaplate-Armand, L. Ferrari, C. Masson,
G. Siest, H. Lambert, S. Visvikis, and A.M. Batt
MP51
Influence of methamphetamine on activity of CYP
2D1 in the isolated perfused Wistar albino rat livers
J. Slíva
MP52
Expression of aryl hydrocarbon receptor repressor
in normal human tissues and inducibility by polycyclic aromatic hydrocarbons in human tumor-derived cell lines
Y. Tsuchiya, M. Nakajima, S. Itoh, M. Iwanari, and
T. Yokoi
MP60
Functional characterization of a CYP3A7 variant
with a longer C-terminal
C. Rodríguez-Antona, M. Hidestrand, A. Rane,
and M. Ingelman-Sundberg
MP61
Effects of weaning to diets containing rice protein
isolate (RPI) on growth, plasma IGF1 and expression of CYP2C11 and CYP4A1 in rat liver
M. J. J. Ronis, M. Reeves, H. Hardy, J. Badeaux,
C. Dahl, D. Harisson, R. Haley, L. Humphrey,
M. Ferguson, and T. M. Badger
MP53
Regulation of mutation process induced by ionizing
radiation in myelocariocytes of mice by peroxidase
and extract from Armoracia rusticana L.
R. Agabeyli, T. Gasimova, and U. Alekperov
MP54
Mouse cytochromes P450 4F
L. Antonovic, X. Cui, H. Kawashima, and
H. W. Strobel
MP62
Effects of weaning to diets containing soy protein
isolate (SPI) or isoflavones on growth, plasma IGF1
and expression of CYP2C11 and CYP4A1 in rat liver
M. J. J. Ronis, M. Reeves, H. Hardy, J. Badeaux,
C. Dahl, D. Harisson, R. Haley, L. Humphrey,
M. Ferguson, and T. M. Badger
MP55
TCDD induces CYP2A5 enzyme in mouse primary
hepatocytes
S. Arpiainen, O. Pelkonen, and J. Hakkola
MP63
Human cryopreserved hepatocytes as an in vitro
model to study cytochrome P450 induction
D. Roymans, P. Annaert, J. Van Houdt, A. Weygers, D. Jacobs, J. Hendrickx, G. Mannens, and
W. Meuldermans
MP56
CYP2C8, CYP2C9, CYP3A7, PXR and CAR mRNA
level increase in hepatoblastoma cell line HepG2, after HMG-CoA reductase inhibitors treatment
C. Bertrand-Thiebault, L. Ferrari, C. Masson,
S. Visvikis, and A. M. Batt
MP64
CYP1A regulation by α-tocopherol
Y. A. Sidorova, and A. Y. Grishanova
MP57
Characterization of cytochrome P450 expression
and induction profiles in the WIF-B9 cell line
F. Borde, V. E. Bender, S. Chevalier, C. Charuel,
D. Cassio, and C. P. Biagini
MP65
The role of cytochromes P450 in biotransformation
of new antileukotrienic drug quinlukast in rat
B. Szotáková, L. Skálová, L. ·i‰pera, and V. Wsól
MP58
The decrease of rat liver microsomal CYP3A2 catalytic activity during aging may be the consequence of an enhanced protein instability
V. Wauthier, R. K. Verbeeck, and P. B. Calderon
MP66
Effect of repeated albendazole administration to
mouflons on albendazole metabolism in vivo and in
vitro
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J. Velík, L. Skálová, V. Baliharová, B. Szotáková,
V. Wsól, and J. Lamka
cine and cytochromes P450 dictates its pharmacological efficiencies
M. Stiborová, J. Sejbal, D. Aimová, T. Dlouhá,
L. Bofiek-Dohalská, K. Forsterová, J. Poljaková,
M. Zachová, M. Stiborová-Rupertová,
K. Kukaãková, A. Bfiezinová, J. Kuãka, and E. Frei
MP67
Age related changes in the protein and mRNA levels of CYP2E1, CYP3A1, CYP3A2 as well as in
their hepatic activities in Wistar rats
V. Wauthier, N. Maton, V. Allaeys, R. K. Verbeeck;
and P. B. Calderon
TP07
Non-aromatic hydroxylation of bupivacaine in man
A. Kumar, B. Hofte, R. Ledger, and U. Tjaden
MP68
Administration of CYP19 modulators to Gallus domesticus
T. Koblas, P. Trefil, M. Stiborová, and P. Hodek
TP08
Inhibition of P450 enzymes participating in p-nitrophenol hydroxylation by drugs known as CYP2E1
inhibitors
K. Monostory, E. Hazai, and L. Vereczkey
Tu e s d a y, J u l y 1
TP09
CYP19 activity modulation: small change of flavonoid structure results in inhibitor-stimulator transition
D. Provazníková, P. Hodek, S. Smrãek,
B. I‰tvánková, and M. Stiborová
TP01
Non-dissociative sequential metabolism of an
8-alkyl xanthine by human P450 1A2
Jason Boer, Kyle E. Allen, and Kent L. K. Kunze
TP10
Potent mechanism-based inhibition of human
CYP2B6 by clopidogrel and ticlopidine
T. Richter, K. Klein, T. E. Mürdter, M. Schwab,
M. Eichelbaum, and U. M. Zanger
TP02
Caffeine oxidation during prolonged exposure of
rats to phenothiazine neuroleptics
W. A. Daniel, M. Kot, and J. Wójcikowski
TP03
Activity of NADPH-cytochrome P-450 reductase in
the human heart, liver and lungs in the presence of
padma 28 - in vitro study
J. Dudka, M. Murias, F. Burdan, J. Szumi∏∏o,
R. Klepacz , and J. Jodynis-Liebert
TP11
CYP17 catalytic iron-oxygen species dictated by
cytochrome b5
P. Lee-Robichaud, Q. Sheikh, and M. Akhtar
TP12
Faradaic bioelectorchemistry for the drug metabolism assay
V. V. Shumyantseva, T. V. Bulko, and A. I. Archakov
TP04
Ethanol influence on methanol oxidation via ADH
and MEOS
K. Dawidek-Pietryka, J. Dudka, and K. Domaƒska
TP13
Structural and functional characterizations of putidaredoxin-induced structural changes in cytochrome P450cam
T. Tosha, S. Nagano, J. K. Yano, S. Yoshioka,
H. Harada, S. Takahashi, K. Ishimori,
T. L. Poulos, and I. Morishima
TP05
Activation of NADPH-cytochrome P-450 reductase
in the human heart, liver and lungs by Ukrain - in
vitro study
J. Dudka, M. Murias, F. Burdan, J. Szumi∏∏o,
R. Klepacz , and J. Jodynis-Liebert
TP14
Chiral aspects of reduction metabolism of flobufen
in guinea pigs in vivo
TP06
The interaction between the anticancer drug ellipti-
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01 Úvod 10.6.2003 11:41 Stránka 17
F. Trejtnar, R. Král, V. Wsól, P. Pávek, and
E.Kvasniãková
TP23
Reduction of oxyferrous cytochrome P450 2B4 by
cytochrome P450 reductase
H. Zhang, L. Gruenke, D. Arscott, A. Shen,
C. Kasper, and L. Waskell
TP15
Multiple P450 substrates in single run: rapid, comprehensive and clinically relevant in vitro metabolism test
M. Turpeinen, J. Uusitalo, J. Jalonen, and
O. Pelkonen
TP24
Human hepatic oxidation of dothiepin mediated by
CYP2D6
H. Yin, P. Tran, and V. Fischer
TP16
+
Immobilisation of P450 BM-3 and an NADP cofactor recycling system: towards a technical application
S. C. Maurer, R. D. Schmid, and V. Urlacher
TP25
Selective dehydrogenated intermediates are mechanism-based inactivators of CYP3A4, CYP2E1,
AND CYP2F1
K. W. Skordos, S. J. Smeal, C. A. Reilly,
D. L. Lanza, and G. S. Yost
TP17
Heterologous expression of full-length NADH-cytochrome b5 reductase and its fusion protein with cytochrome b5
A. A. Gilep, V. V. Kokhanovski, and S. A. Usanov
TP26
Human and minipig cytochrome P450 2E1
E. Anzenbacherová, J. Baranová, R. Zuber,
P. Anzenbacher, and P. Souãek
TP18
Overexpression of full-length outer mitochondrial
cytochrome b5 in E. coli
A. A. Gilep, G. V. Sergeev, A. Ito, and
S. A. Usanov
TP27
Two novel human P450 enzymes, CYP4Z1 and
CYP4X1
N. J. Horley, T. Jiang, and D. R. Bell
TP19
Engineering of proteolytically stable NADPH-cytochrome P450 reductase
A. A. Gilep, T. A. Bonina, R. W. Estabrook, and
S. A. Usanov
TP28
Polymorphism of P450 in Drosophila melanogaster
A. Brun-Barale, D. Crochard, S. Tares, L. Arthaud,
J.-M. Bride, and M. Amichot
TP20
C-terminal residues of cytochrome P450scc are required for correct folding, heme binding and catalysis
G. I. Lepesheva, N. V. Strushkevich, T. N. Azeva,
and S. A. Usanov
TP29
Bacterial monooxygenases in biotransformation
and biodegradation of aliphatic and aromatic compounds
V. Leont’ev, T. Achramovitch, I. Burak, and
O. Ignatovets
TP21
Spectroscopic studies of cytochrome P4503A4 interaction with flavonoids
M. V. Chudaev, E. M. Tcherviakovsky,
V. P. Kurchenko, and S. A. Usanov
TP30
Rabbit as an animal model to study CYP3A-mediated drug-drug interactions
N. Chauret, L. Sleno, J. Silva, R. Houle, S. Day,
and D. Nicoll-Griffith
TP22
Human cytochrome P450 2D6-catalyzed reactions:
The kinetic interaction between two substrates
R. W. Wang, X. Cai, V. Didolkar, R. W. Edom,
R. Evers, and D. C. Evans
TP31
CYP2D6 polymorphism and extrapyramidal symptoms in patients on long term antipsychotic treatment
17
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V. DolÏan, B. Kores-Plesniãar, B. Zalar, and
K. Breskvar
TP40
Transcript analysis and azole inhibition of mycobacterial CYPs
J. Parker, C. Jackson, T. Marczylo, S. Perally,
D. Lamb, S. Kelly, and D. Kelly
TP32
CYP2U1, a thymus and cerebellum expressed cytochrome P450 converts arachidonic acid into bioactive derivatives 19- and 20-HETE
C. Helvig, S. Chuang, M. Taimi, G. Jones,
M. Petkovich, J. A. White, and B. Korczak
TP41
CYP 2D6‚ intermediate metabolizer’ phenotype as
a consequence of genetically determined alternative splicing
C. Cesaro, S. Raimundo, K. Klein, E. Schaeffeler,
M. Eichelbaum, M. Schwab, and U. M. Zanger
TP33
A novel CYP26 family member metabolizes 9-cisretinoic acid
M. Taimi, C. Helvig, M. Petkovich, and B. Korczak
TP42
Identification of CYP2D6 genotype via a new sequencing protocol
J. K. Coller, H. M. James, D. Gillis, B. C. Sallustio,
and A. A. Somogyi
TP34
cDNA cloning and biochemical characterization of
CYP4F11, a novel human cytochrome P450
C. Helvig, and B. Korczak
TP35
CYP2C9 polymorphism and warfarin dose requirement
D. Herman, T. Globokar, P. Peternel, M. Stegnar,
K. Breskvar,V. DolÏan
TP43
Use of 2D-electrophoresis and MALDI-TOF in the
identification of hepatic CYP1A isoforms of Prochilodus scrofa, a Brazilian freshwater fish
M.E.F. Da Silva, J. A. Silva, D. Martins,
S. Marangoni, J.C. Novello, and N. C. Meirelles
TP36
A streptomyces CYP105D1 exported in Escherichia
coli induces periplasmic accumulation of porphyrin
M. K. Akhtar, N. N. Kaderbhai, and M. A. Kaderbhai
TP44
NF-I regulates CYP2A5 transcription in mouse primary hepatocytes
J. Ulvila, O. Pelkonen, M. Negishi, and J. Hakkola
TP37
Novel SNP’s in the CYP2B6 gene and their functional consequences
K. Klein, T. Lang, T. Richter, M. Eichelbaum,
M. Schwab, and U. M. Zanger
TP45
CYP2C9 phenotyping using low-dose tolbutamide
A. Jetter, M. Kinzig-Schippers, A. Skott, A. Lazar,
D. Tomalik-Scharte, M. Walchner-Bonjean,
U. Hering, H. Lück, W. Jabrane, D. Kasel,
U. Fuhr, and F. Sörgel
TP38
Comparative analysis of CYP3A expression in human liver suggests only a minor role for CYP3A5 in
drug metabolism
S. Malmebo, A. Westlind-Johnsson, A. Johansson, C. Otter, T. B. Andersson, I. Johansson,
R. J. Edwards, A. R. Boobis, and
M. Ingelman-Sundberg
TP46
A novel thymus-specific cytochrome P450, CYP2U1
M. Karlgren, and M. Ingelman-Sundberg
TP47
Rapid translocation of CYP11A1 into yeast mitochondria hampers its normal sorting and folding
I. E. Kovaleva, L. A. Novikova, P. A. Nazarov,
S. I. Grivennikov, and V. N. Luzikov
TP39
Searching for the gene encoding steroid 11β-hydroxylase of the filamentous fungus Cochliobolus lunatus
E. Ku‰ãer, L. Lah, and R. Komel
TP48
Synthesis and properties of fused proteins compo-
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01 Úvod 10.6.2003 11:41 Stránka 19
sed of components of the cholesterol side-chain
cleavage enzyme system
P. A. Nazarov, V. L. Drutsa, W. L. Miller,
V. M. Shkumatov, V. N. Luzikov, and L. A. Novikova
the rat CYP2B2 phenobarbital response unit:
HNF4 and Pbx-Prep1
M.-J. Beaudet, and A. Anderson
TP57
Rabbit CYP4B1 engineered for high-level expression in E. coli: ligand stabilization and processing
of the N-terminus and heme prosthetic group
M. J. Cheesman, B. R. Baer, E. M. J. Gillam, and
A. E. Rettie
TP49
P450 allele and haplotype frequency study in diffe™
rent populations using the CODELINK SNP assay
R. Bonner, B. Chui, M. Gaskin, P. Gwynne,
A. Ledesma, T. Peck, T. Peters, A. Silbergleit,
M. Amjadi, R. Feldman, and C.Yamashiro
TP58
Transcriptional regulation of the cholesterol biosynthetic pathway in the male gonad
K. Fon Tacer, D. Rozman
TP50
Association of genetic polymorphisms in cytochrome P450 2E1 and other biotransformation enzymes with progression of lymphomas
P. Souãek, J. ·armanová, S. ·Ûsová, and I. Gut
TP59
Constitutive androstane receptor-retinoic x receptor (CAR-RXR)-dependent transcriptional activation by the CYP2B2 phenobarbital response unit
varies dramatically with different reporter constructs
M. Gravel, M. Desrochers, É. Trottier, and A. Anderson
TP51
Genome-wide expression profiling and xenobiotic
inducibility of P450 monooxygenase genes in the
white rot fungus Phanerochaete chrysosporium
J. S. Yadav, and H. Doddapaneni
TP52
Haplotype analysis of cytochrome P450 2B6 - linkage between promoter- and exonic mutations
J. Zukunft, T. Lang, K. Klein, T. Richter,
M. Schwab, M. Eichelbaum, and U. M. Zanger
TP60
Expression of mammalian microsomal P450s in
plants using plant virus vectors
A. A. Jgoun, M. A. Eldarov, N. A. Petushkova,
V. K Novikov, Y. L. Dorokhov, I. G. Atabekov, and
K. G. Skryabin,
TP53
High level expression of porcine P450 reductase
solubilized doman in Escherichia coli by modulating the local secondary structure of mRNA
Shigenobu Kimura, and Takashi Iyanagi
TP61
Expression of leukotriene B4 ω-hydroxylase in human leukocyte
H. Kunishi, Y. Masumoto, M. Eya, J. Ohta,
S. Nishimoto, K. Nakata, and Y. Kikuta
TP54
Interaction of constitutive androstane receptor-retinoic x receptor (CAR-RXR) heterodimers with elements of the CYP2B2 phenobarbital response unit
M.-J. Beaudet, A. Lachaud, and A. Anderson
TP62
NR3 and ER-7 nuclear receptor sites of the
CYP2B2 phenobarbital response unit: mutational
analysis of their importance for PB-dependent
transcriptional activation
M. Laizier, Y. Paquet, A. Anderson
TP55
The anticancer drug ellipticine acts as an inducer
of cytochromes P450 1A1/2 and potentiates its
own pharmacological efficiency
D. Aimová, T. Dlouhá, E. Frei, and M. Stiborová
TP63
Characterization of cytochrome P-450 26 (CYP26)
enzymatic activity and comparison with other CYPs
involved in all-trans-retinoic acid (ATRA) metabolism
J. Marill , A. Picard-Cloutier, and G. G. Chabot
TP56
Newly-identified transcription factor binding sites in
19
01 Úvod 10.6.2003 11:41 Stránka 20
TP64
Distribution of glutathione S-transferases in mullet
(Liza saliens)
A. Kirikbakan, and A. Sen
WP02
Aflatoxin B1 binding to mutant Phe209Ala CYP2A5
expressing Saccharomyces cerevisae yeast cells
P. Bartakova, H. El-Nezami, H. Raunio, and
R. O. Juvonen
TP65
Biochemical, molecular genetic and environmental
aspects of mullet (Liza saliens) CYP1A1
A. Sen, A. Kirikbakan, D. R. Buhler, and E. Arinc
WP03
Engineering substrate recognition in catalysis by
cytochrome P450cam
S. G. Bell, X. Chen, F. Xu, Z. Rao, and L.-L. Wong
TP66
Albendazole induced CYP1A activities in mouflons
(Ovis musimon)
L. Skálová, V. Baliharová, J. Velík, B. Szotáková,
V. Wsól, and J. Lamka
WP04
Validation of commercially supplied cryopreserved
hepatocytes from a range of species for use in metabolic profiling studies
C. Dilworth, R. Shannly-Henderson, V. Eagling,
P. D. Fenn, and G. R. Murray
TP67
Cytochrome P450 3A4 expression is reduced in
the presence of cancer-associated inflammation in
a humanised CYP3A4 transgenic mouse model
K. Slaviero, A. Lehnert, S. Bierach, C. Liddle,
S. Clarke, L. Rivory, G. Robertson
WP05
P450 2E1/BMR: studies of a human/bacterial P450
fusion protein
M. J. Fairhead, and G. Gilardi
TP68
Activity of cytochromes P450 2C in rat cardiomyocytes
L. Kousalová, J. Baranová, J. Psotová,
E. Anzenbacherová, and P. Anzenbacher
WP06
O2 metabolism by soluble cytochrome P450 reductase and its chimeras with C-termini of nitric oxide
synthases assayed by diacetyldeuteroheme HRP
Y. Ishimura, S. P. Panda, M. Jáchymová,
P. Martásek, T. Kitazume, J. A. Peterson,
L. J. Roman, and B. S. S. Masters
TP69
Metabolism of alpha-naphthoflavone and its thioanalogue by microsomal system
P. Valentová, P. Hodek, L. Bofiek-Dohalská,
M. ·ulc, and M. Stiborová
WP07
An homogeneous assay for screening CYP2D6 inhibitors using scintillation proximity assay technology
E. Beynon, M. Price-Jones, J. Berry, and
K. T. Hughes
TP70
Application of α-naphthoflavone during the growth
and development of broilers
A. Miãáková, P. Hodek, M. Popl‰tein and P. Trefil
WP08
Exploration of natural and artificial P450 sequence
spaces
P. Urban, V. Abécassis, G. Truan, and D. Pompon
WP09
Cytochrome P4502E1 activity and free radical oxidation in the liver of hypothyroid rats with acute alcohol intoxication
N. Kaloshyna, T. Khomich, and P. Pronko
We d n e s d a y, J u l y 2
WP01
Molecular breeding of cytochrome P450 enzymes
involved in xenobiotic metabolism
N. Rosic, T.G.A. Lonhienne, J.J. DeVoss, and
E. M. J.Gillam
WP10
Assignment of omeprazole-sensitivity gene locus,
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01 Úvod 10.6.2003 11:41 Stránka 21
whose product mediates CYP1A1 induction, on human chromosome 10
H. Kikuchi, S. Fukushige, M. Shibazaki, A. Sohel,
and T. Takeuchi
(NAT2) phenotypes as markers of cancer susceptibility by workers exposed to aromatic amines
J.. Pastera, P. Anzenbacher, Z. Fiala,
R. Nádvorníková, J. ·alandová, and J. Kvûtina
WP11
Study on organ specificity of CYP1A1 induction by
1-phenylazo-2-hydroxynaphthalene (Sudan I) in rats
V. Marková, V. Martínek, P. Hodek, and
M. Stiborová
WP19
Method of preliminary selection of substances specific in relation to P450 isoforms
A. A. Pogrebnoy, V. E. Ryabinin, V. A. Potemkin,
E. V. Bartashevich, and M. A. Grishina
WP12
The use of bacterial membranes in the study of interactions of human cytochromes P450 and xenobiotics
E. Kondrová, and P. Souãek
WP20
Peroxidase and CYP-mediated ellipticine-DNA adduct
formation explains the selective efficiency of this anticancer drug against breast cancer and leukemia
J. Poljaková, K. Forsterová, M. ·ulc, E. Frei, and
M. Stiborová
WP13
Azole antifungal drugs act upon cytochrome P450
mono-oxygenases to efficiently inhibit growth in
Mycobacteria and Streptomycetes
K. R. Marshall, K. J. McLean, D. Leys, and
A. W. Munro
WP21
Distinct inhibition of human CYP2A6 and mouse
CYP2A5 by derivatives of lactone, phenylethylamine and benzaldehyde
M. Rahnasto, H. Raunio, A. Poso, and
R. O. Juvonen
WP14
Sudan I metabolism by human cytochrome P450
1A1 is stimulated by microsomal cytochrome b5
V. Martínek, and M. Stiborová
WP22
Characterization of human P450 involved in oxidation of manidipine, a 1,4 dihydropyridine calcium
channel blocker
B. Riccardi, P. Puccini, D. Spinabelli, and D. Acerbi
WP15
P450 aromatase inhibition assay based on an
ELISA system
Kazuhiro Matsui, Shigeaki Nishii, Takuya Ishibashi,
and Masanori Oka
WP23
Human cytochromes P450 1A1/2 and NADPH:
cytochrome P450 reductase activate carcinogenic
aristolochic acid to form DNA adducts found in
patients with Chinese herbs nephropathy
M. Stiborová, E. Frei, B. Sopko, K. Sopková,
V. Marková, M. LaÀková, T. Kumst˘fiová,
M. Wiessler, and H. H. Schmeiser
WP16
Cytochrome P450-mediated activation of carcinogenic 2-anisidine leads to formation of DNA adducts in vitro and in vivo
M. Mik‰anová, L. Vondrá‰ková, M. ·ulc,
H. H. Schmeiser, E. Frei, and M. Stiborová
WP24
Interaction of omeprazole and caffeine on cyto13
chrome P450 1A2 evaluated using C-caffeine
breath test in rats
Z. Svoboda, J. Kvûtina, J. Bure‰, and V. Vofií‰ek
WP17
Possible role of CYP450s in the nabumetone
metabolism
M.Nobilis, E.Anzenbacherová, M.Holãapek,
L. Koláfiová, J. Kvûtina, and P. Anzenbacher
WP25
Microsomal transformation of organophosphorus
pesticides by white rot fungi
R. Vazquez-Duhalt, and J. Jauregui
WP18
Cytochrome P450 1A2 and N-acetyltransferase
21
01 Úvod 10.6.2003 11:41 Stránka 22
WP26
Development of an in vitro reporter system to assess the transcriptional regulation of the human
CYP1A2 gene
F. Amin, M. Town, A. R. Boobis, and D. S. Davies
Yu. D. Ivanov, I. P. Kanaeva, I. I. Karuzina,
V.Yu. Kuznetsov, S. A. Usanov, G. Hui Bon Hoa,
S. G. Sligar, and A. I. Archakov
WP35
Metabolism of N-benzyl-1-aminobenzotriazole by
P450 2B1 results in a modified enzyme capable of
N-demethylation but with abolished hydroxylation
activity
U. M. Kent, R. A. Roof, L. Pascual, D. P. Ballou,
and P. F. Hollenberg
WP27
SAR-by-NMR with CYP450eryF: implications for
homotrophic/heterotrophic cooperativity
J. N. Lampe, J. T. Pearson, J. S. Grinstead,
M. J. Dabrowski, A. P. Campbell, and W. M. Atkins
WP28
Improving the affinity of adrenodoxin for its partner
proteins by directed evolution
A. Bichet, F. Hannemann, and R. Bernhardt
WP36
A proton-shuttle mechanism mediated by the porphyrin in benzene hydroxylation by cytochrome
P450 enzymes
S. P. de Visser, and S. Shaik
WP29
Novel reversible adduction of the P450 heme: inactivation of cytochrome P450 2E1 T303A by tertbutyl acetylene
A. L. Blobaum, U. M. Kent, and P. F. Hollenberg
WP37
Effect of memantine on in vitro cytochrome P450mediated metabolic activities in human liver microsomes
S. Micuda, L. Mundlova, E. Anzenbacherova,
P. Anzenbacher, J. Chladek, J. Martinkova
WP30
Protein engineering of human cytochrome P450s
for structure determination
V. Dodhia, R. Bazin, and G. Gilardi
WP38
21-fluorosteroids used in kinetic and 19F-NMR studies to probe CYP17 catalysis
E. O’Donnell, J. N. Wright, and P. Lee-Robichaud
WP31
Studies on interaction between fibrates and statins
H. Fujino, S. Shimada, I. Yamada, M. Hirano,
Y. Tsunenari, and J. Kojima
WP39
Effects of thalidomide on the CYP-mediated metabolism of cyclophosphamide in the mouse
J. Paxton, P. Kestell, Q. Ding, L.-M. Ching,
B. D. Palmer, S. Zhou, and B. C. Baguley
WP32
The effects of chronic treatment with antidepressant drugs on the level and activity of CYP2C6,
CYP2C11 and CYP3A1/2 in the rat liver
A. Haduch, and W. A. Daniel
WP40
Reduction of the nitroaromatic antiandrogen nilutamide by NO synthases. Implications for the adverse effects of this antiandrogen drug
S. Dijols, K. Ask, C. Giroud, L. Casse, Y. Frapart,
M.-A. Sari, P. Camus, D. Stuehr, D. Mansuy, and
J.-L. Boucher
WP33
The optical biosensor study of redox partners’ interactions in the P4502B4 system in hydroxylation
conditions
Yu.D. Ivanov, V.Yu. Kuznetsov, N.A. Petushkova,
I.P. Kanaeva, and A.I Archakov
WP41
Dynamics of CYP2D6 activity and clinical response
to paroxetine, alprazolam and their combination in
patients with depressive disorder
E. Hada‰ová, A. Îourková, J. Tomandl, and
B. Ravãuková
WP34
Atomic force microscopy (AFM) and optical biosensor studies of cytochrome P450’s component
interactions
22
01 Úvod 10.6.2003 11:41 Stránka 23
WP42
Inhibition of spontaneously produced nitric oxide in
conventional and perfused hepatocyte culture
improves cell functionality
N. Canová, E. Kmoníãková, and H. Farghali
synthase expression following keratoplasty in
mice
P. Stfie‰tíková, J. Pl‰ková, J. Martínek, M. Filipec,
and H. Farghali
WP43
Differences and comparisons of the active species
(Cpd I) of cytochrome P450 and horse radish peroxidase as revealed by density functional theoretical studies
S. P. de Visser, P. K. Sharma, D. Kumar, and S. Shaik
WP51
Mechanistic studies on the intramolecular oneelectron transfer between the two flavins in iNOS
flavin domain and NADPH-cytochrome P450 reductase
K. Yamamoto, Z.-W. Guan, Y. Nishino, S. Kimura,
and T. Iyanagi
WP44
Inhibition of inducible nitric oxide synthase activity
by oltipraz in vitro and in activated microglia cells
L. L. Furge, P. R. Fields, W. E. Goode,
M. C. Tressler, and R. S.-Truss
WP52
Xanthates (dithiocarbonates) metabolism by some
monooxygenases
S. Yanev, B. Pandova, T. A. Vannelli, V. Todorova,
and P. R. Ortiz de Montellano
WP45
Endothelial nitric oxide synthase reductase domain
is activated by calmodulin and phosphorylation
Y. Nishino, K. Yamamoto, Z.-W. Guan, S. Kimura,
and T. Iyanagi
WP53
The metabolic ratio carbamazepine/carbamazepine-epoxide in children and adult patients on antiepileptic polytherapy
B. Brzakovic, M. Pokrajac, Î. Martinovic,
B. Miljkovic, R. Velickovic
WP46
Recruitment of governing elements for electron
transfer in nitric oxide synthase family
M. Jáchymová, P. Martásek, S. Panda, T. M. Shea,
L. J. Roman, and B. S. S. Masters
WP54
New in vitro metabolites of paclitaxel in humans,
rats, minipigs and regular pigs and CYP involved in
their formation
R. Václavíková, S. Horsk˘, and I. Gut
WP47
Modulation of electron transfer in nitric oxide synthase using non amino acid guanidines and hydroxyguanidines
M. Moreau, J.-L. Boucher, S. Dijols, Y. Frapart,
D. Lefevre, D. Stuehr, and D. Mansuy
WP55
Quantitative evaluation of relative contribution of
human cytochrome P-450 isoenzymes (CYPs) to
the metabolism of promazine
J. Wójcikowski, W.A. Daniel, and
L. Pichard-Garcia, P. Maurel
WP48
On the role of the cysteine ligand in cytochromes
P450
P. Rydberg, E. Sigfridsson, and U. Ryde
WP56
Stimulation of human cytochrome P450 3A4 activity by anionic phospholipids and their micro
-domains
K.-H. Kim, T. Ahn, and C.-H. Yun
WP49
Is NOS a NOx-Synthase?
J. Santolini, Z.-Q. Wang, D. Konas, and
D. J. Stuehr
WP57
Potent inhibition of human cytochrome P450
-catalyzed reactions by hemin
E.-Y. Kim, J.-S. Kim, M.-Y. Kim, W.-S. Koh,
F. P. Guengerich, and C.-H.Yun
WP50
Induction and modulation of inducible nitric oxide
23
01 Úvod 10.6.2003 11:41 Stránka 24
WP58
Genetic polymorphisms in human CYP2A6 gene
and interindividual differences in nicotine metabolism
M. Nakajima, R. Yoshida, K. Nishimura,
J.-T. Kwon, and T. Yokoi
I. Manzanares, G. Mannens, W. Meuldermans,
and R. De Coster
WP67
Differences in biotransformation of testosterone by
brain microsomes of rat and rabbit: a preliminary
study
L. Marvasi, P. Anfossi, and A. Zaghini
WP59
Genetic polymorphism of canine cytochrome P450
2D15 gene
I. S. Pappas, and D. S. Katsiabas
WP68
Polymorphism in cytochrome P450 2D6-mediated
activation of malathion in rats
V. Silvari, S. Catania. A. Palamara, L. B. Martino,
K. Zahlsen, and C. Costa
WP60
The involvement of cytochrome P450 in the metabolism of chloroquine on human liver microsomes
K.-A. Kim, J.-Y. Park, J.-S. Lee, and S. Lim
WP69
In vitro studies on the activation of the organophosphate pesticide malathion by cytochrome P450
2D6 in rat liver microsomes
C. Costa, S. Catania, V. D’Angelo, L. B. Martino,
K. Zahlsen, and V. Silvari
WP61
Inhibition of cytochrome P450 activities by oleanolic acid and ursolic acid in human liver microsomes
K.-A. Kim, J.-S. Lee, H.-J. Park, C.-J. Kim,
I.-S. Shim, S.-M. Han, J.-W. Kim, and S. Lim
WP70
Photoaffinity labeling of cytochrome P450 2B4
M. Karabec, M. ·ulc, and P. Hodek
WP62
Cloning, sequencing and heterologous expression
of the genes coding for the P450mor system from
Mycobacterium sp. strain HE5
B. Sielaff, and J. R. Andreesen
WP63
Coumarin 7-hydroxylase activity in liver and brain
microsomes of mice
E. D. Ozdamar, M. Iscan, and B. C. Eice
WP64
CYP4F8 and CYP4F12: tissue distribution and catalytic properties
K. Stark, L. Schauer, J. Bylund, and E. H. Oliw
WP65
Baboon CYP11B1: the expression and structural
analysis of two catalytically active isoforms
A. C. Swart, S. Graham, N. Brown, and P. Swart
WP66
An interspecies comparison of the metabolism
™
of the anticancer agent Yondelis (trabectedin,
ET-743)
M. Vermeir, J. Hendrickx, R. Hurkmans, J. Van
Houdt, C. Zwijsen, N. Bode, P. Aviles,
24
01 Úvod 10.6.2003 11:41 Stránka 25
GENERAL INFORMATION
CONFERENCE VENUE
Národní dÛm na Vinohradech (National House of Vinohrady)
Námûstí Míru 9
120 53 Prague 2
Phone: +420-221 596 311, 221 596 111
Fax: +420-221 596 234
(Metro station „Námûstí Míru“ - line A)
All events of the Scientific Program are realized in this building. As the halls and rooms are mostly located at the same floor, there is no need to present a layout scheme of the building here. The lecture halls
as well as the space allocated for posters and for exhibition are indicated by arrows and signs.
BADGES
Participants will be given a badge upon registration. The badge must be worn at all times; only registered participants will be allowed to enter the
Conference area.
●
●
●
●
●
●
REGISTRATION DESK
The registration desk, located at the National House
of Vinohrady (Conference Venue), will be opened for
the duration of the Conference as follows:
Sunday, June 29, 2003
Monday, June 30, 2003
Tuesday, July 1, 2003
Wednesday, July 2 , 2003
Thursday, July 3, 2003
12.00
7.30
8.00
8.00
8.00
-
Coffee/tea service
Get-Together Party (Sunday, June 29, 2003)
Opening Ceremony (Monday, June 30, 2003)
Concert (Monday, June 30, 2003)
3 lunches
Free ticket for Prague metropolitan transport
valid for 5 days
Accompanying persons
On-site registration / paid after APRIL 30, 2003
140 USD
20.00
17.00
14.00
16.00
13.00
Registration fee covers:
● Admission to exhibition area
● Get-Together Party (Sunday, June 29, 2003)
● Opening Ceremony (Monday, June 30, 2003)
● Concert (Monday, June 30, 2003)
● City Tour of Prague
● Free ticket for Prague metropolitan transport
valid for 5 days
REGISTRATION FEES
Full participants
On-site registration / paid after APRIL 30, 2003
560 USD
CERTIFICATE OF PARTICIPATION
The certificate of participation will be issued upon
request to properly registered participants. The
Certificate will be available at the registration desk
on the last day of the Conference.
Full registration fee covers:
● Admission to all scientific sessions and exhibition area
● Conference documents (program, book of abstracts, list of participants)
● Certificate of participation
LUNCHES AND REFRESHMENT
Coffee or tea will be provided during the coffee
breaks to all registered participants in the Exhibi-
25
01 Úvod 10.6.2003 11:41 Stránka 26
tion/Refreshment Hall. Packed lunches will be served to all registered participants in exchange for
the respective ticket at distribution points.
SOCIAL PROGRAM
The following social program will be prepared for
the participants of the Conference. The additional
tickets for Get-Together Party and Concert can be
bought by the registration desk.
EXHIBITION
The exhibition is located at the Exhibition/Refreshment Hall just in front of the main lecture hall
(Mayakoffsky Hall).
CURRENCY
The official currency is the Czech Crown (CZK =
Kã). Exchange rate fluctuates daily, but at the time
of printing this Program one USD was equivalent to
approximately 26 CZK.
1) GET-TOGETHER PARTY
One ticket included in the registration fee
Sunday, June 29, 19.00-21.00
Additional ticket: 300 CZK
INSURANCE, SECURITY
The Conference Secretariat will not accept any liability for personal injuries, loss or damage to property of participants and accompanying persons.
Kindly take your personal insurance policy. The
site of the Conference is under security control. As
Prague is attracting not only many tourists, but
also dishonest people, please, take care of your
personal belongings.
National House of Vinohrady
- Conference Venue (Námûstí Míru 9, Prague 2)
On Sunday, June 29, 2003, the participants are invited to the Mayakoffsky Hall, the most representative space of the National House. Welcome drink
and small refreshment will be available, transfer
will not be provided.
2) CONCERT
One ticket included in the registration fee
Monday, June 30, 20.00-21.00
Additional ticket: 300 CZK
CLIMATE AND DRESS
The weather at the end of June and beginning of
July is usually pleasant with an average day temperature of 20-25 °C. Informal dress will be suitable for the Conference and social events.
St. Simon and Jude Church (Du‰ní 1, Prague 1)
Connection from the Conference Venue: by
metro A from „Námûstí Míru“ to „MÛstek“, 2 stops
and by walk (approximately 30 minutes). The concert of ensemble „ Musica Gaudeans“ (Joyful Music) will take place at the concert hall situated at
the baroque Church of St. Simon and Jude. The
ensemble consisting of flute, oboe, guitar and cello
will present the Conference participants an interesting program (Bach, Vivaldi, Paganini, Lobos).
Transfer will not be provided.
TRANSPORTATION
Metropolitan transportation in Prague is quite reliable and consists of trams, buses and metro (subway).
Metro is the most reliable and the most popular
way of transport in Prague. Metro system consists
of three lines - A, B, C, connecting suburban areas
with the city center. On each metro train there is a
map of lines with detailed description.
At the Registration Desk each participant and
accompanying person will obtain a free ticket
to Prague metropolitan transport (metro, buses, trams). The ticket is valid for 5 days (incl.
the first day). Please stamp the ticket in the
stamping machine when entering either the
tram, the bus or at the entrance to the subway
(Metro).
3) STEAMBOAT PARTY
Tuesday, July 1, 16.00 -19.00
35 USD
Meeting point: steamer landing-place (between
Palackého and JiráskÛv bridges) at 15.45 hrs.
Connection to the meeting place from the
26
01 Úvod 10.6.2003 11:41 Stránka 27
Conference Venue: by tram No. 4 or No. 16 from
„Námûstí Míru“ to „Palackého námûstí“, 5 stops (approximately 20 minutes). During this afternoon trip on
board of the biggest Prague steamer you will have an
opportunity to see the most attractive sights in Prague.
Enjoy a romantic afternoon with a buffet dinner and
live swing music. Transfer to and from the steamer
landing-place will be realized by public transportation.
4) FAREWELL DINNER
Wednesday, July 2, 19.00 - 24.00
Sightseeing tour will take you to Prague Castle and
some parts of Old Town and other historical city
quarters. The bus will depart from the Conference
venue. It will stop at the Castle area, which you will
explore on foot. This is the biggest castle area in
the world, still in use as a seat of the Presidents of
the Czech Republic. The price covers the admission fee to St. Vitus Cathedral, Old Royal Palace
and Golden Lane.
For registered accompanying persons one City
tour is free of charge. It is advisable to book
a seat in advance and choose the date of tour.
Without the preliminary booking we cannot guarantee your seat.
65 USD
Trip to the Mûlník Castle with wine tasting, dinner and program
Meeting point: at the Theatre Vinohradske divadlo just near by the Conference Venue.
Time: 18.40, meeting in front of the theatre,
kindly note that the buses will depart from the theatre in 10 min. intervals. The last one will depart
at 19.00! Please, do not be late!
2) JEWISH GHETTO + OLD TOWN SQUARE
Tuesday, July 1, 10.00 - 14.30
USD 42
The bus will take you from the Conference Venue
to the Old Town Jewish quarter. You will visit its
amazing synagogues incl. the oldest preserved synagogue in Europe and the Jewish Cemetery from
XV century with its 1200 fascinating tombstones
and mystic atmosphere. The famous Rabbi Löw,
creator of the legendary Golem is resting here as
well as numerous other personalities. At the end
you will walk to the nearby Old Town Square with
the popular Horologe presenting a moving show of
12 apostles. The bus will take you back to the Conference Venue or you can stay at the Old Town and
enjoy its beauties on your own.
Enjoy the atmosphere of the Renaissance castle
near Prague. You will visit the historical interior and
taste the wine from the cellars of the Lobkowicz
noble family. Afterwards you will take part in a festival with knight tournament, Gothic and Renaissance dances while tasting the rich buffet dinner
and drink the castle wines and famous Czech beer.
Departure from Mûlník Castle: 23.00
Arrival to Prague: 24.00
3) TRIP TO CASTLE KARL·TEJN & NIÎBOR
GLASS FACTORY
Wednesday, July 2, 9.00 - 17.30
65 USD
TOURIST PROGRAM
Meeting point for all trips is at the registration
desk at the Conference venue. At the trip counter
you will receive your prepaid tickets and you may
buy tickets for all tours if the place is available.
1) CITY TOUR
Sunday, June 29
Monday, June 30
Wednesday, July 2
You will visit the family glass factory producing famous Czech crystal glass. A small glass souvenir
is included, you will have also an opportunity to
shop in the factory shop. From the factory the route
follows through the romantic forested countryside
to the Karl‰tejn Castle, founded in XIV century as
a treasury for safeguarding the coronation jewels
and holy relics. The castle is the most popular one
in the whole country, except for Prague Castle.
13.00 - 17.00
USD 28
10.00 - 14.00
cancelled due to small
number of participants
27
01 Úvod 10.6.2003 11:41 Stránka 28
Lunch will be served in the village below the castle
in a typical restaurant.
4) TRIP TO KONOPI·Tù CHATEAU
Thursday, July 3, 9.00 - 14.00
been once home of the successor of the Austrian
throne, Franz Ferdinand d’Este, whose assassination in Sarajevo sparked off the World War I. The
chateau offers a large display of historical weapons, hunting trophies, etc. You will see that Mr.
d’Este was really a keen hunter and lover of fine
arts. Beautifully furnished and decorated rooms
will surely attract your eyes. A large English park
surrounds the chateau.
Lunch is not included.
48 USD
This excursion will show you one of the most beautiful chateaux in Bohemia. The chateau has
ACCOMMODATION
The participants who used the
services of Conference Secretariat (CBT) for hotel booking and
who need any further assistance
regarding the room reservation
are asked to kindly contact the
Accommodation desk in the Registration office during the announced registration hours.
For an official invoice or receipt
kindly contact the Accommodation desk, the relevant document will be issued on request.
28
01 Úvod 10.6.2003 11:41 Stránka 29
TH
13
INTERNATIONAL CONFERENCE
ON
CYTOCHROMES P450
Biochemistry, Biophysics and Drug Metabolism
ABSTRACTS
Prepared for print by
Jifií Hudeãek
Editing:
The documents presented in this Volume were not changed by the Organizers and
are presented as submitted by the Authors, except for adjustment of formatting and
for corrections of the occasional obvious typing errors in the titles of the contributions. Therefore, the Authors bear the sole responsibility for the scientific content of
all abstracts.
Disclaimer
The published contributions present the views of their respective Authors and the
content is the sole responsibility of the Authors. The mere fact that they have been
published in this Volume does not imply their endorsement or liability by the Organizers or by the supporting and sponsoring bodies and organizations (as e.g. the
European Commission, IUBMB and others). In no case the Organizers or the Sponsors should be held responsible for any loss or damage caused by the use of the
data appearing in this publication.
29
01 Úvod 10.6.2003 11:41 Stránka 30
02 LectML.QXD 10.6.2003 12:10 Stránka 31
Chem. Listy, Symposia, 97 (2003)
1
Invited Lectures – ML
dues, and the mutants were purified from an E. coli
expression system and assayed for progesterone hydroxylation. Based on the results, a quadruple mutant
I114A/F206V/F297G/V363L, termed Q, was constructed
that showed 60% of 2C5 progesterone 21-hydroxylase
activity and 57% regioselectivity. Based on their 2C5-like
testosterone hydroxylation profiles, S294D and I477F alone
and in combination were added to the quadruple mutant.
All three mutants showed enhanced regioselectivity (70%)
for progesterone 21-hydroxylation, while only Q-I477F had
a higher kcat. Finally, V103I was added to Q-I477F and
Q-S294D/I477F. Q-V103I/S294D/I477F showed a 3-fold
higher kcat than 2C5 and 80% regioselectivity for progesterone 21-hydroxylation. Docking of progesterone into
a 3D model of this mutant indicates that 21-hydroxylation
is favored. In conclusion, a systematic approach to convert
P450 regioselectivity across subfamilies suggests that active site residues are mainly responsible for regioselectivity differences between 2B1 and 2C5, and validates the
reliability of 2B1 models based on the crystal structure of
2C53.
LECTURES – MONDAY – ML
ML01 STRUCTURAL BASIS OF P450
2B SPECIFICITY
JAMES R. HALPERT, SANTOSH KUMAR,
EMILY E. SCOTT, and YOU QUN HE
Department of Pharmacology & Toxicology, University of
Texas Medical Branch, 301 University Blvd., Galveston,
Texas 77555-1031, USA, [email protected]
Introduction
The long-term objective of our research is to elucidate
the structural basis for the substrate specificities of cytochromes P450 of the 2B subfamily. P450 2B enzymes are
very versatile catalysts with a broad range of substrates,
including drugs, environmental carcinogens, and steroids.
Along with members of the 2A and 2C subfamilies, P450
2B enzymes are thought to exhibit the least degree of catalytic preservation across mammalian species. This observation suggests a strong role for steric constraints imposed
by the enzymes, as opposed to the inherent chemical reactivity of the compounds, in determining substrate specificity. Through studies during the past decade the key amino
acid residues that dictate ligand binding orientation within
the interior of the active site and contribute to substrate and
inhibitor specificity of several P450 2B enzymes have been
identified1. All these residues have counterparts in the active site of P450 2C5 inferred from the x-ray crystal structure2. Such information has allowed us to explain many of
the functional differences among P450 2B enzymes and to
begin to alter their activities in a rational manner. However,
the promise of engineering novel or more efficient catalysts of specific oxidative reactions remains largely unmet,
and the pathways by which different substrates gain access
to the buried active site remain to be clarified. Our central
hypothesis is that substrate specificity reflects the interplay
between amino acid residues in the interior of the active
site and those that line the substrate access channel.
A Rational Approach to Re-engineer Cytochrome P450
2B1 Regioselectivity Based on the Crystal Structure of
P450 2C5. Interest in structure-function studies across
P450 subfamilies was sparked by the considerable effort in
a number of laboratories to use the P450 2C5 structure to
model other mammalian P450 enzymes and predict their
substrate specificities and stereo- and regioselectivity. An
implicit assumption in all such models based on a single
template is that the backbones of the enzymes are essentially invariant and that active site differences alone are responsible for specificity differences. Therefore, we sought
to confer the progesterone hydroxylation specificity of
P450 2C5 on 2B1. P450 2B1 is a high Km progesterone
16-hydroxylase, whereas 2C5 is a low Km progesterone
21-hydroxylase. Initially, nine individual P450 2B1 active
site residues were changed to the corresponding 2C5 resi-
Substrate access channel
Substrate interactions with cytochromes P450 have
been proposed to occur in three stages:
1) recognition of the substrate by surface residues;
2) entry into the buried active site through a hydrophobic access channel;
3) substrate orientation in the active site to allow catalysis. Until recently, all known structures of bacterial cytochromes P450 suggested that substrate access to the buried
active site occurred via the F-G region, a surface loop distal
to the heme cavity. However, the structure of P450 51 indicates a large opening from the protein surface along the
I helix N-terminus, at right angles to the F-G channel. The
single available microsomal P450 structure (2C5) does not
obviously favor one potential access route over the other. To
determine whether the F-G region forms part of the substrate access channel in cytochrome P450 2B1, eleven residues between positions 208 and 230 were substituted with
smaller and larger side chains in a highly expressed truncated form of the enzyme. Steady-state kinetic parameters
were determined with the substrates testosterone, 7-ethoxy4-trifluoromethylcoumarin (7-EFC), and 7-benzyoxyresorufin (7-BR). The largest changes, 2 to 6-fold increases in
kcat with testosterone and 7-EFC, were observed for L209A,
which also exhibits an altered testosterone metabolite profile and probably forms part of the active site roof.
F219W demonstrated little or no activity with any of the three substrates examined, although the Ks value for benzphetamine binding was unaltered. S221F showed little activity
with 7-BR. No significant changes were observed in Km
(testosterone) or S50 (7-EFC) values for any of the mutants,
in stark contrast to the ten-fold and hundred-fold changes in
Km observed for mutants in this region of other cytochromes
P4504. The minimal changes in 2B1 did not support access
via the F-G region of 2B1 and suggested the alternate
S31
access route identified in P450 51. More recent unpublished
studies suggest a role for residues in the N-terminal part of
the I helix in regulating the efficiency of substrate hydroxylation, consistent with a role in substrate access.
a highly purified enzyme, P450 2B4 has long served as
a model for biophysical studies of mammalian cytochromes
P450. Thus, structures of P450 2B4 have the potential to integrate decades of functional and mechanistic analysis.
Acknowledgment
Supported by NIH grants ES03619 (JRH), GM20674
(EES) and Center Grant ES06676.
Methods
Cytochrome P450 2B4 was engineered to remove the Nterminal transmembrane helix, substitute a number of charged residues at the new N-terminus, and add four histidine residues to the C-terminus. The resultant E. coli-expressed protein (2B4dH) was bound to membranes, but could be liberated by high salt conditions. Although the protein could be purified in the absence of detergents, the crystallized protein
was extracted from membranes and stabilized during Ni2+-affinity and CM-Sepharose chromatography with detergent.
Analytical ultracentrifugation indicated that protein purified
in the presence of sodium cholate was a monodisperse monomer, while purification in the presence of Cymal-5 yielded
two species, presumably a monomer and a dimer.
Crystals were initially grown by equilibrating a concentrated protein solution including 30 mg/ml P450 and
4.8 mM Cymal-5 with 15% ethanol and 0.1 M sodium citrate, pH 5.5 using sitting drop vapor diffusion at 18° C.
Macroseeding allowed the growth of single crystals with
an increase in size and thickness, resulting in individual
crystals up to 1 mm in length. Crystals were briefly flooded with mother liquor containing 30% glyercol and immediately frozen in liquid N2 at 100 K.
Data analyzed for the final structure were collected at the
Stanford Synchrotron Radiation Laboratory from a single
crystal. Crystals diffracted to 1.6 Å. The space group is
C2221 with cell dimensions a=57.09 Å, b=114.20 Å,
c=133.95 Å and a single molecule in the asymmetric unit
(solvent content ~40%). The structure of 2B4 was solved
by molecular replacement using a model of 2C5/3LVdH
(1N6B) with the non-identical residues mutated to Ala.
CNS was used for early rounds of refinement and SHELX
for later rounds of refinement.
REFERENCES
1. Domanski, T.L., Halpert, J.R.: Curr. Drug Metab. 2, 117
(2001).
2. Williams, P.A., Cosme, J., Sridhar, V., Johnson, E.F.,
McRee, D.E.: Mol. Cell 5, 121 (2000).
3. Kumar, S., Scott, E.E., Liu, H., Halpert, J.R.: J. Biol.
Chem., in press (2003).
4. Scott, E.E., He, Y.Q., Halpert, J.R.: Chem. Res. Toxicol. 15,
1407 (2002).
ML02 A 1.6 Å CRYSTAL STRUCTURE
OF P450 2B4: NOVEL FEATURES
AND IMPLICATIONS
EMILY E. SCOTTa, YOU AI HEa,
MICHAEL R. WESTERb, MARK A. WHITEc,
JAMES R. HALPERTa, ERIC F. JOHNSONb, and
CHARLES D. STOUTd
a
Department of Pharmacology and Toxicology, University
of Texas Medical Branch, 301 University Blvd., Galveston,
Texas 77551-1031, USA, [email protected], bDepartment
of Molecular and Experimental Medicine MEM-255, The
Scripps Research Institute, 10550 N. Torrey Pines Rd., La
Jolla, CA 92037, USA, cX-ray Crystallography Core, Sealy
Center for Structural Biology, University of Texas Medical
Branch, 301 University Blvd., Galveston, TX 77551-0647,
USA, dDepartment of Molecular Biology MB-8, The
Scripps Research Institute, 10550 N. Torrey Pines Rd.,
La Jolla, CA 92037, USA.
Structure of P450 2B4.
The secondary structure and general tertiary structure
of P450 2B4 conform to the known P450 architecture, consisting of a beta sheet domain and an alpha helix domain.
As expected, the structure of P450 2B4 shares more structural similarity with P450 2C5 than with the bacterial enzymes, but significant conformational differences were observed in the helix F-helix G region, the helix B-helix C region, and helix I relative to the 2C5 structure.
The F-G region is composed of helix F, two short helical segments (F’ and G’), and helix G. The placement and
structure of helix F is similar to that in 2C5 except that the
C-terminal turn is unwound. The conformation of helix
G is largely conserved, but it rises at a sharper angle above
the active site than in 2C5. The F’ and G’ helices and the
N-terminus of helix G extend away from the core of the
protein, resulting in the formation of a large open cleft between the F-G region and the B’- C loop. This cleft extends
Introduction
Cytochromes P450 from families 1, 2, and 3 are among
the most versatile biological catalysts known. Each enzyme
is responsible for the biotransformation of an array of structurally variable xenobiotics and endogenous compounds,
with overlapping specificities among enzymes. Understanding the structure-function relationships of these monooxygenases has been significantly enhanced by the structure of
the first membrane P450, 2C51. We have proposed that the
versatility of substrate oxidation among the xenobiotic-metabolizing P450 enzymes is likely to be reflected in their
structural diversity. We present here the first structure of
a P450 from the 2B subfamily. Due to its availability as
S32
directly from the protein surface to the heme iron. The intermolecular packing is such that the extended F’- G’ structural motif of one molecule fills the open cleft of a symmetry-related molecule. In the crystal, a dimer is formed by
a bond between His226 at the extreme terminus of the F’G’ extension in one molecule and the heme iron of a molecule related by the crystallographic 2-fold axis. Spectral
data indicate that this dimer forms reversibly in solution.
The opposing side of the cleft is formed primarily by the
loop between helices B’ and C and the C helix, the positions
of which are altered relative to 2C5. The flexible loop between the helices B’ and C demonstrates some of the highest
B values in the structure. Corresponding to these conformational differences the propionate side chain of the heme ring
D interacts with Arg98, found before the helix B’, and with
Arg133, found in the C terminus of helix C, instead of Trp120
and Arg124, both in the N terminus of helix C, in 2C5.
Finally, while helix I of 2C5 is relatively straight, 2B4
has a distinct bend in helix I. When the two structures are
overlapped using the conserved tertiary structure, the Ntermini of the I helices differ by 2.8 Å.
Although the possibility cannot be excluded that intermolecular contacts influence the structure of the F-G region in P450 2B4dH, crystallization of flexible protein regions generally appears to select an individual conformation from the continuum present in solution. The differences between the structure of P450 2C5, and the present
structure of P450 2B4 suggest the possibility that the FG and B’-C regions of family 2 enzymes may adopt a substantial range of energetically accessible conformations.
CYP2C9 is expressed at high level in human liver and is
the enzyme responsible for the metabolism of more than
10% of the drugs currently in clinical use. Functional polymorphism of CYP2C9 also has clinical implications in
drug-drug interactions and in variation in individual response to therapeutic drugs. To date, information on the topology of the CYP2C9 active site has derived from predictive models based on the bacterial and the rabbit CYP2C5
crystal structures, combined with site directed mutagenesis
studies. However, the low amino acid sequence identity
(20-30%) shared with the bacterial templates and the medium resolution in the CYP2C5 structure of regions that
control the topology of the active site and the substrate recognition, has yielded unsatisfactory models for the prediction of the mode of binding of compounds in the active
site. We have determined a 2.6 Å x-ray structure of a soluble form of CYP2C9. The crystal structure of CYP2C9
provides a basis for the accurate identification and localization of the key residues that constitute the active site and
better understanding of the mechanisms that lead to differences in substrate specificities.
ML04 STRUCTURAL STUDIES OF
SUBSTRATE BINDING TO
CYP2C DRUG METABOLIZING
P450S
ERIC F. JOHNSONa, MICHAEL R. WESTERa,
GUILLAUME SCHOCHa, JASON K. YANOa, and
CHARLES D. STOUTb
Acknowledgment
Supported by NIH grants GM20674 (EES), ES03619
(JRH), GM31001 (EFJ), GM59229 (CDS), and the Sealy
and Smith Foundation (MAW).
a
Department of Molecular and Experimental Medicine
MEM-255, The Scripps Research Institute, 10550 N. Torrey
Pines Rd., La Jolla, CA 92037, USA, bDepartment of Molecular Biology MB-8, The Scripps Research Institute,
10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA,
[email protected].
REFERENCES
1. Williams P.A., Cosme J., Sridhar V., Johnson E.F.,
McRee D.E.: Mol. Cell 5, 121 (2000).
Comparative analysis of the mouse and human genomes indicates that genes encoding family 2 P450s exhibit
some of the highest rates of non-synonymous nucleotide
substitutions in these genomes.1 This genetic diversity underlies a functional diversity that is particularly striking for
family 2C P450s within and between species and is amplified by the capacity of these enzymes to recognize and metabolize structurally diverse substrates. Human 2C8, 2C9
and 2C19 contribute significantly to the metabolism of
drugs used therapeutically, and genetic polymorphisms and
metabolic drug-drug interactions involving these enzymes
can lead to adverse drug effects. The structures of CYP
2C5, 2C8, and 2C9 illustrate a number of factors that contribute to the substrate selectivity and the catalytic diversity of these enzymes.
The structures of the CYP 2C proteins exhibit general
features seen for soluble, bacterial P450s. The most signi-
ML03 INSIGHTS ON THE TOPOLOGY
OF THE ACTIVE SITE OF
HUMAN CYTOCHROME P450
2C9 DETERMINED BY X-RAY
CRYSTALLOGRAPHY
JOSE COSME, PAMELA WILLIAMS, ALISON WARD,
DIJANA VINKOVIC and HARREN JHOTI
Astex Technology Ltd, 436 Cambridge Science Park, Cambridge CB4 0QA,UK [email protected]
Cytochrome P450 2C9 (CYP2C9) ranks amongst the
most important drug-metabolizing enzyme in humans.
S33
ficant differences from soluble P450s are the presence of
a longer polypeptide chain between helix F and helix
G that contains two short helices, F’ and G’ and an extended N-terminal amino acid sequence that includes a hydrophobic transmembrane helix (residues 1-20) that is connected by a polar linker to a proline rich motif (residues 3037) at the beginning of the catalytic domain. The N-terminal domain has been deleted in all three proteins to facilitate crystallization, and a 4 histidine tag was added to the
C-terminus to facilitate purification. In addition, CYP 2C5
is mutated at 5 residues on helix F and in the turn preceding
helix F’ to facilitate crystallization.2
The structures of the three CYP 2C proteins are highly
similar, but differ in features that contribute to substrate
binding. Although these include differences in the amino
acid side chains that can potentially contact substrates in
the substrate-binding cavity, differences are also evident in
the positioning of the peptide backbone. Differences in the
conformation of the peptide backbone are greatest for helix
B to helix C region and from helix E through the N-terminal portion of helix I. The flexibility of these regions also
allows some degree of expansion or contraction to adapt to
substrates of different sizes. As a result of differences in
conformation and amino acid side-chains, the active site
volume of 2C8 is roughly twice that of 2C5.
Several factors are likely to contribute to substrate binding. These include steric fit, van der Waals and hydrogen
bonding interactions and the hydrophobic effect. The active site cavities are significantly larger than the space
occupied by some substrates. This can give rise to multiple binding orientations for the substrate that can lead to
alternative products. In general, both the substrates and
the active site cavities are largely hydrophobic, and elimination of water from the hydrophobic surfaces of the substrate and the active site cavity will contribute significantly to the free energy of substrate binding. Residual
water molecules that are not displaced by the substrate
contribute to hydrogen bonding interactions between the
polar moieties of the substrate and the protein, and these
waters can mask unfavorable electrostatic interactions.
Polar interactions contribute significantly to substrate binding and the regioselectivity of metabolism. This is clearly
illustrated by the structures of CYP 2C5 and 2C9 with
nonsteroidal anti-inflammatory drugs bound in their active
sites.
ML 05 RADICALS AND STRESSED
BACTERIAL P450 ENZYMES
WESLEY STRAUB, and
PAUL R. ORTIZ DE MONTELLANO
Department of Pharmaceutical Chemistry, University of
California, 600 16th Street, San Francisco, CA 941432280, USA, [email protected]
Replacement of the heme iron atom has been achieved
with many hemoproteins, but only limited success has been
reported in this area for cytochrome P450 enzymes. Cytochrome P450cam has been reconstituted with other metal
porphyrins, notably Co and Mn, but reconstitution requires
fairly harsh conditions that can perturb the native metal environment.1 We report here a biosynthetic approach to the
assembly of metal substituted cytochrome P450 enzymes
based on heterologous expression of the proteins in E. coli
under controlled metal conditions. The approach thus far
has been used to prepare cobalt-substituted cytochrome
P450cam (CYP101) and CYP119 with no more than trace
amounts of the iron porphyrin. Both of these proteins possess a lower affinity for the Co-PPIX prosthetic group than
for heme and are less thermally stable. X-ray crystal structures of the two proteins suggest that these properties are
due to a longer metal-sulfur bond. Earlier crystal structures
of CYP119 with imidazole and 1-phenylimidazole coordinated to the iron atom demonstrated the presence of two distinct protein conformers.2 2D NMR studies of cobalt
CYP119 without added ligands confirm the existence of at
least two equilibrating protein conformers whose populations are altered upon ligand binding. Cobalt CYP119 has
been found to catalyze the hydroxylation of lauric acid in
the presence of NADPH and a surrogate electron donor
system, but not in the presence of H2O2. The different reactivity profiles of the iron and cobalt proteins may reflect
subtle differences in their hydroxylation mechanisms. In
contrast, cobalt CYP101 reacts quantitatively with peroxides to yield novel porphyrin adducts that decompose to
stable cobalt hydroxyporphyrins. The ability to prepare otherwise unperturbed metal-substituted P450 enzymes promises to open a new area of catalytic chemistry involving
thiolate-ligated metalloprotein active sites. This work was
supported by NIH Grant GM25515.
Acknowledgment
Supported by NIH grants GM31001 (EFJ) and
GM59229 (CDS). Facilities for DNA sequencing and oligonucleotide synthesis are supported in part by the Sam
and Rose Stein Charitable Trust.
REFERENCES
1. Wagner, G. C., Gunsalus, I. C., Wang, M. Y., Hoffman,
B. M.: J. Biol. Chem. 256, 6266 (1981).
2. Yano, J. K., Koo, L. S., Schuller, D. J., Li, H., Ortiz de
Montellano, P. R., Poulos, T. L.: J. Biol. Chem., 275,
31086 (2000).
REFERENCES
1. Waterston, R. H. et al. Nature 420, 520 (2002)
2. Cosme, J. and Johnson, E. F. J.Biol.Chem. 275, 2545
(2000).
S34
large quantities using a heterologous expression system.
One step in the biosynthesis of epothilones involves a 1213 epoxidation (Fig. 1) by a cytochrome P450, P450epoK.
The structures of P450epoK in the substrate-free, substrate-bound, and product bound complexes have been solved to a resolution of 2.65 Å, 1.93 Å, and 2.10 Å, respectively. As expected, P450epoK most closely resembles
P450eryF, which also participates in the biosynthesis of
a complex macrolide. The active site region of the substrate complex is shown in Fig. 2. Similar to other P450s,
the atoms that undergo the epoxidation reaction are close to
the heme iron and in a position to directly contact the Felinked O atom. The thiazole portion of the substrate provides key contact points which very likely are critical in substrate specificity. Interestingly, there are strong parallels to
the binding of epothilones to P450epoK and Taxol to tubulin. An unexpected outcome of these structures is that there
is little difference in the substrate-bound and -free structures. We had anticipated a more open conformation of the
substrate access channel in the substrate-free structure but
there is only a slight closure upon substrate binding. Nevertheless, there must be a fairly large open/close motion
in order to accommodate such a large substrate.
ML06 STRUCTURES OF P450EPOK,
PUTIDAREDOXIN, AND
PUTIDAREDOXIN REDUCTASE
IRINA SEVRIOUKOVAa, SHINGO NAGANOa,
HUIYING LIa, CLINTON NISHIDAb,
HIROSHI OGURAb,
PAUL R. ORTIZ DE MONTELLANOb,
and THOMAS L. POULOSa
a
Department of Molecular Biology and Biochemistry,
Department of Physiology and Biophysics, and Program in
Macromolecular Structure, University of California, Irvine,
California 92697-3900, USA. bDepartment of Pharmaceutical Chemistry, University of California, San Francisco,
California 94143-2280, USA.
▲
Epothilones (Fig.1) are naturally produced by the cellulose-degrading myxobacterium Sorangium cellulosum
strain So ce90. Like the well-known anti-cancer agent, Taxol, epothilones bind to and stabilize microtubles leading
to mitotic arrest of the cycle at G2-M phase and subsequ-
Fig. 1. Reaction catalyzed by
P450epoK.
▲
▼ Fig. 2. Two views of the P450epoksubstrate complex. Ala250 in the I helix
and Phe96 provide key contact points
with the thiazole part of the substrate.
The arrows indicate the carbon atoms
which form the epoxide.
Fig. 3. Crystal structures of Pdx and Pdr.
ently induces apoptosis in several cell lines. Epothilones
offer some advantages since epothilones are effective against P-glycoprotein-expressing multi-drug resistant cell lines, are active in a cell line with taxol resistance, and epothilones water solubility is significantly greater than Taxol.
Another advantage is that epothilones can be produced in
Structures of the electron transfer components of the
P450cam monooxygenase system, putidaredoxin (Pdx) and
putidaredoxin reductase (Pdr), have been solved to a resolution of 1.75 Å and 1.9 Å, respectively. A comparison
with mammalian counterparts, adrenodoxin (Adx) and adrenodoxin reductase (Adr), reveals both similarities and
S35
differences. The Pdx and Adx (1) structures clearly are
very similar. However, Pdr and Adr (2) exhibit large differences. The FAD site is less exposed in Pdr owing to an
additional β-pair situated near the cofactor. In addition, the
C-terminal region of Pdr is positioned very differently than
in Adr. Therefore, if Pdx and Pdr form a complex similar
to the Adr-Adx complex, then the C-terminal domain in
Pdr must move. This seems unlikely since the C-terminal
region that would be required to move is composed of
β-sheets that tie this region to the main body of the protein.
Therefore, Pdx forms a complex with Pdr that must be quite different than the Adx-Adr complex. The implications
of these structures for protein-protein recognition and electron transfer will be described.
a series of rearrangements before deprotonation to form
many different cyclic and acyclic skeletal structures. These
parent hydrocarbons, and their oxygenated derivatives
such as the alcohols, epoxides, aldehydes and ketones, are
the major fragrance and flavouring components in plant essential oils. For example, carveol, perillyl alchol and isopiperitenol derived from the oxidation of limonene,1,2 and
verbenol, verbenone and myrtenol obtained from α-pinene
oxidation, are fine chemicals.
We have investigated the oxidation of limonene and pinene by cytochrome P450cam (CYP101) from Pseudomonas
putida.3-5 The bicyclic (+)-α-pinene is structurally related
to camphor, the natural substrate of P450cam. The enzymesubstrate contacts in P450cam has been investigated extensively by biophysical methods as well as crystallography.4,6
Based on this wealth of information and comparison of the
structure of camphor and pinene, we introduced a number
of active site mutations to compensate for the structural
differences. We have shown that a small number of mutations can enhance the monoterpene oxidation activity of
P450cam. The effect of mutations on selectivity is complicated and sometimes non-additive. The crystal structure of
a key mutant (F87W/Y96F/V247L) with (+)-α-pinene
bound within the active site was determined to provide insights into the enzyme-substrate interactions.7 The (+)-αpinene substrate is bound in two different orientations in
the enzyme. One of these is closely related to that of camphor in the wild type enzyme, while the other orientation
can be generated by a simple rotation from the first orientation. The structure allows new mutations to be introduced
to alter the selectivity of product formation.7
REFERENCES
1. Müller, A., Müller, J. J., Muller, Y. A., Uhlmann, H.,
Bernhardt, R., and Heinemann, U. Structure 6, 269
(1998)
2. Ziegler, G. A., Schulz, G. E. Biochemistry 39, 10986
(2000).
ML07 CRYSTAL STRUCTURE-BASED
ENGINEERING OF SUBSTRATE
RECOGNITION IN P450CAM
CATALYSIS
STEPHEN G. BELLa, XUEHUI CHENb,
REBECCA J. SOWDENa, FENG XUb,
ZIHE RAOb, and LUET-LOK WONGa
REFERENCES
1 Colby S.M., Alonso W.R., Katahira E.J., McGarvey
D.J., Croteau R., J. Biol. Chem. 268, 23016 (1993).
2 Lupien S., Karp F., Wildung M., Croteau R., Arch.
Biochem. Biophys. 368, 181 (1999).
3 Gunsalus I. C., Wagner G. C., Methods Enzymol.8 52,
166 (1978).
4 Mueller E. J., Loida P. J., Sligar S. G. ‘Twenty-five years of P450 cam research. Mechanistic insights into
oxygenase catalysis’, in Cytochrome P450: Mechanism,
Structure and Biochemistry 2nd edn. (Ortiz de Montellano P. R., ed.), New York 1995.
5 Bell S. G., Sowden R. J., Wong L.-L., Chem. Commun.
635 (2001).
6 Poulos T. L., Cupp-Vickery J., Li H., ‘Structural studies
on prokaryotic cytochromes P450’, in Cytochrome
P450: Mechanism, Structure and Biochemistry 2nd edn.
(Ortiz de Montellano P. R., ed.) New York 1995.
7 Bell S. G., Chen X., Sowden R. J., Xu F., Williams J.
N., Wong L. L., Rao Z., J. Am. Chem. Soc. 125, 705
(2003).
a
Department of Chemistry, Inorganic Chemistry Laboratory, South Parks Road, Oxford OX1 3QR, UK; bLaboratory of Structural Biology and Protein Science, Tsinghua
University, Beijing 100084, China;
[email protected].
The C-H bond oxidation activity, under ambient conditions, of P450 enzymes has no equivalent in classical synthetic methodology and thus has great potential in applications such as fine chemical synthesis. For such processes to
be viable the enzymes must have high activity and selectivity for the oxidation of unnatural substrates, and ideally it
should be possible to alter the selectivity for the synthesis
of isomeric compounds, including enantiomers. At the heart of this lies the challenge of understanding and ultimately manipulating the enzyme-substrate molecular recognition.
The terpenoid hydrocarbons (C5H8)n are biosynthesised
by the coupling of isoprenyl units. The monoterpenes
(n=2) are derived from geranyl pyrophosphate. Terpene
synthases catalyse the dissociation of the pyrophosphate
group, and the carbonium ion thus generated undergoes
S36
This laboratory entered into the nitric oxide synthase
(NOS) field as a result of the high sequence homology between NADPH-cytochrome P450 oxidoreductase (CYPOR)
and the 641 C-terminal residues of neuronal NOS1 and the
finding that this enzyme was a flavoprotein containing one
mol each of FAD and FMN per monomer2, 3. Subsequent sequence comparisons have led to the conclusion that the differences between the simpler CYPOR structure and the
more complex NOS isoforms have resulted from the evolution of these proteins to become modulated in order to
participate in cell signaling processes. The formation of
NO in cellular systems, by necessity, must be carefully regulated and localized.
Comparison of the NOS and CYPOR sequences identified autoregulatory inserts in the FMN-binding domains of
both constitutive isoforms, neuronal (nNOS) and endothelial NOS (eNOS), absent in CYPOR and the inducible NOS
isoform, that inhibit NOS activity until Ca+2/calmodulin
(CaM) is bound4. Furthermore, all three NOS isoforms
contain C-terminal sequences that extend beyond the C-terminus of CYPOR at the identical residue in each isoform.
This laboratory5,6 demonstrated, by truncating the three isoforms to resemble CYPOR, that the additional C-terminal
sequences act as inhibitory modulators of NOS activity. In
all three isoforms, this truncation resulted in 7- to 10-fold
increases in flavoprotein-mediated cytochrome c reduction
that were reduced to the wild type activity upon the addition of Ca2+/calmodulin. NO production by these truncated
mutants is either only slightly affected in the case of inducible NOS, which contains tightly bound CaM, or is decreased by 45% and 33% in nNOS and eNOS, respectively,
upon the addition of CaM. These results indicate an interactive relationship between the autoregulatory inserts and
CaM binding in the activation of nNOS and eNOS.
Recent experiments (Roman et al. (2003) in preparation) have determined which structural domains are involved in the regulation of catalysis of both electron transfer
from the flavoprotein domain and the production of NO
through the heme domain by neuronal NOS. Chimeric constructs were expressed in which the FAD- and/or FMN-binding domains of CYPOR were swapped with the respective
nNOS domain to determine the role of the flavin-binding
subdomains in catalyzing these activities. Constructs of
nNOS containing the FAD-binding domain of CYPOR catalyzed high rates of cytochrome c reduction (>7000 min-1
vs. 2500 min-1 for wild type) in the absence of CaM. Cytochrome c reduction was inhibited by the addition of CaM
to the construct in which the FMN-binding domain was derived from nNOS and the FAD-binding domain was from
CYPOR, similar to the C-terminal tail-less mutant of
nNOS. The chimera, containing both flavin-binding domains of CYPOR, catalyzed a high rate of cytochrome c reduction that was unaffected by the addition of CaM. Ferricyanide reduction was not affected by the addition of CaM
in any of the constructs. These results indicate the effects
of CaM are primarily mediated through the FMN-binding
domain of nNOS, since ferricyanide reduction occurs
ML08 STRUCTURAL ASPECTS
OF LIGAND BINDING TO
PROCARYOTIC AND FUNGAL
P450S
OLENA PYLYPENKOa,b, KATJA ZERBEc,
FRANCESCA VITALIc, WEIWEN ZHANGc,
SEVERINE ROUSETTc, MARCUS HECKc,
JAN W. VRIJBLOEDc, DANIEL BISCHOFFd,
BOJAN BISTERd, RODERICH D. SÜSSMUTHd,
STEFAN PELZERe, WOLFGANG WOHLLEBENe,
JOHN A. ROBINSONc, and ILME SCHLICHTINGa,b
a
Max Planck Institute for Molecular Physiology, Dept. of
Physical Biochemistry, Otto Hahn Str.11, 44227 Dortmund,
Germany; bMax Planck Institute for Medical Research,
Dept. of Biomolecular Mechanisms, Jahnstr. 29, 69120 Heidelberg, Germany; cInstitute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich,
Switzerland; dInstitut für Organische Chemie, EberhardKarls-Universität Tübingen, Auf der Morgenstelle 18,
72076 Tübingen, Germany; eLehrstuhl für Mikrobiologie/Biotechnologie, Eberhard-Karls-Universität Tübingen,
Auf der Morgenstelle 28, 72076 Tübingen, Germany
P450 enzymes are involved in numerous biological
processes including the biosynthesis of lipids, steroids, antibiotics, and the degradation of xenobiotics. In line with
the variety of reactions catalyzed, the size of the substrate
varies significantly. Some P450s have open active sites
(e.g. BM3), some have shielded active sites that open only
transiently (e.g. P450cam), whereas others bind the substrate only when attached to carrier proteins (e.g. oxy proteins). In addition to the organic substrate, oxygen has to
bind. Structural aspects of both organic and gaseous ligand
binding are described and discussed.
ML09 REVELATIONS OF NITRIC
OXIDE SYNTHASE
MODULATION: HIDDEN
MESSAGES IN SEQUENCE
AND STRUCTURE
BETTIE SUE S. MASTERSa, LINDA J. ROMANa,
PAVEL MARTÁSEKb, MARIE JÁCHYMOVÁa,b,
YUZURU ISHIMURAa, SATYA P. PANDAa,
THOMAS M. SHEAa, DAWN E. HARRISa,
and JENNIFER MCLAINa
a
Department of Biochemistry, Univ. of Texas Health Sci. Ctr.
at San Antonio, TX 78229-3900, USA; bThe Inst. of Clin. Biochem. and Department of Pediatrics, Faculty General Hospital and Charles University, Prague 2, Czech Republic
S37
through the FAD-binding domain of both CYPOR and
NOS. The only chimera catalyzing NO formation contained the heme- and FMN-binding domains of nNOS and the
FAD-binding domain of CYPOR and was capable of producing higher amounts of NO than the wild type nNOSa superenzyme.
Chimeric constructs were also made by attaching the
C-terminal tails of each of the three NOS isoforms to soluble CYPOR. In recent experiments (Jachymova et al.
(2003) in preparation), chimeric proteins of CYPOR containing each of these C-termini were spectrally identical
but showed decreasing cytochrome c and DCIP reductase
activities corresponding to the length of the C-terminus added but revealed little effect on ferricyanide reduction. All
three chimeras exhibited significantly increased (1.8-2fold) NADPH oxidase activity. Also, the rate of reoxidation to the air-stable semiquinone state was substantially
increased. These experiments corroborate the role of the Cterminus in regulating NOS activity and show the degree to
which this regulation is mediated by the length of the Cterminal tail explaining, in part, the sluggish rate of eNOS
catalysis, since it bears the longest C-terminus.
Site-directed mutants of the constitutive NOS isoforms
(Panda et al. (2003) in preparation) in which the serine residue homologous to S457 of CYPOR is mutated to a threonine or alanine residue have been produced. These mutations, which have profound effects in CYPOR7, have also
been shown to affect electron transfer in NOS isoforms.
The catalysis of cytochrome c reduction by the Ser to Ala
mutants is inhibited to a large degree in both isoforms and
both mutants unexpectedly showed measurable activity
that was increased upon the addition of CaM. The
S942T eNOS mutant, on the other hand, catalyzed cytochrome c reduction to the same extent as wild type in the
absence of CaM but was stimulated to approximately half
the activity of the wild type by CaM. The nNOS mutants,
S1176A and S1176T, were both inhibited to a large extent
but the addition of CaM produced very low, but measurable, cytochrome c reductase activities. Interestingly, the
catalysis of NO production by nNOS S1176T was unaffected and that of eNOS S942T was stimulated, while the
alanine mutants were both inhibited by 60-70%. As the
hydroxyl group of the serine residue lies within H-bonding
distance of the O4 and N5 positions of the isoalloxazine
ring of FAD in the nNOS structure8, it is interesting to speculate on the effects of these mutations on overall catalysis.
5.
6.
7.
8.
Smith SME, Martasek P, Roman LJ, Masters BSS, Jones CL, Weissman BD, Lane P, Liu Q, Gross SS:J. Biol.
Chem. 272, 29769 (1997) .
Roman LJ, Miller RT, de la Garza MA, Kim J-JP, Masters BSS: J. Biol. Chem. 275, 21914 (2000).
Roman LJ, Martasek P, Miller RT, Harris DE, de la
Garza MA, Shea TM, Kim J-JP, Masters BSS: J. Biol.
Chem. 275, 29225 (2000).
Shen AL, Kasper CB: Biochemistry, 35, 9451 (1996).
Zhang J, Martasek P, Paschke R, Shea T, Masters BSS,
Kim J-JP: J. Biol. Chem., 276, 37506 (2001).
ML10 FORMATION OF NITRIC OXIDE
FROM XENOBIOTICS
DANIEL MANSUY
UMR 8601, Université Paris 5, 45 rue des Saints-Pères,
75270 Paris Cedex 06, France
Several physiopathological situations correspond to
a deficit in the biosynthesis of nitric oxide (NO) in some
tissues or organs. In such circumstances, it is important to
increase the level of NO locally. This is usually performed
by administration of NO donors such as trinitroglycerin,
which are unstable compounds with very low half-lives in
vivo, and which have a very low selectivity for their targets. Another possible approach towards more selective
compounds, that would lead to an increase of NO formation in a given tissue or organ, is to act on the two main enzymes involved in the metabolism of L-arginine, the NOsynthases and the arginases. In that context, a first strategy
was to find exogenous molecules, xenobiotics, that would
act as substrates of NOS with formation of NO. The administration of such molecules should lead to a selective increase of NO formation via a given NOS. A second strategy
was to inhibit the arginases that catalyze the consumption
of L-arginine with the formation of ormithine and urea,
with the hope that the use of arginase inhibitors would locally increase NO formation by increasing the pool of arginine available for NOS.
As far as the first strategy is concerned, it was important to determine the structural factors that are required for
a compound to be recognized by NOS and to be oxidized
by these enzymes with formation of NO. For that purpose,
we have synthesized a great number of N-substituted guanidines and N-hydroxyguanidines, and we have compared
their modes of binding and affinities for NOS I, II and III,
as well as their oxidation by these enzymes1,2. Most guanidines and N-hydroxyguanidines are well recognized by the
NOS active site, with binding constants that may be similar to those of the NOS natural substrates, arginine and Nhydroxyarginine, provided that their substituent is small
enough. However, only few of them are efficiently oxidi-
REFERENCES
1. Bredt DS, Hwang PM, Glatt CE, Lowenstein C, Reed
RR, Snyder SH: Nature 351, 714 (1991).
2. Hevel JM, White KA, Marletta MA: J. Biol. Chem. 266,
22789 (1991).
3. Stuehr DJ, Cho HJ, Kwon NS, Weise MF, Nathan CF:
Proc. Natl. Acad. Sci., USA 88, 7773 (1991).
4. Salerno JC, Harris DE, Irizarry K, Patel B, Morales AJ,
S38
ML11 TRACKING REACTIVE
INTERMEDIATE STATES OF
NO SYNTHASE UNDER
EXTREME CONDITIONS OF
TEMPERATURE AND PRESSURE
zed by NOS with formation of NO. This is particularly
true for N-substituted guanidines, whose oxidation is more
difficult than that of the corresponding N-hydroxyguanidines, as we only found so far very few exogenous guanidines acting as good substrates for NOS II. The best one,
a fluorinated guanidine, is oxidized by NOS with formation of NO with a Vm value only 3-times lower than arginine itself 2. This reaction not only occurs with recombinant NOS II but also in activated macrophages. The most
active NOS substrates exhibit two characteristics : (i) they
are well recognized by NOS active sites, and (ii) they markedly increase the rate of NADPH consumption by NOS.
These properties are necessary but not sufficient, and supplementary properties appear to be required for obtaining
a good NOS substrate.
Arginases, that catalyze the hydrolysis of L-arginine to
ornithine and urea, are proteins involving a binuclear Mn
(II) active site. We have synthesized powerful inhibitors of
arginases, starting from the 3D structure of their active site.
The most active inhibitors involved a N-hydroxyguanidine
or amidoxine function and an (-aminoacid function separated by two or three atoms3. X-ray studies of several arginase-inhibitor complexes showed that the terminal (N)-OH
group of these compounds acts as a bridging ligand of the
Mn (II) atoms4, after displacement of the H2O bridging ligand that is present in free arginase. Preliminary experiments indicate that such arginase inhibitors are able to increase NO formation in physiopathological situations involving a deficit of NO.
STEPHANE MARCHALa, TON GORRENb,
MORTEN SOERLIEc, KRISTOFFER ANDERSSONd,
BERND MAYERb, and REINHARD LANGEa
a
INSERM U128, 1919 route de Mende, 34293 Montpellier,
France; bInstitut für Pharmakologie und Toxikologie, KarlFranzens-Universität, Universitätsplatz 2, 8010 Graz,
Austria; cAgricultural University of Norway, IKB, NLH,
1432 As, Norway; dDepartment of Biochemistry, University
of Oslo, PO Box 1041 Blindern, N-0316 Oslo.
The catalytic step of nitric oxide synthase (NOS), i.e.,
activation of oxygen and product formation, shows similarities, but also striking differences to the analogous P450
reaction. Especially, it involves the participation of an additional electron donor, tetrahydrobiopterin (BH4). The reaction is complex and fast, comprising elusive intermediates. Subzero temperature in the liquid state, and high pressure can be used as perturbing parameters to study this
complex reaction step by step, and to characterize structurally intermediate states of the reaction. We used these
techniques to study both NOS reaction cycles and to study
specifically the second electron transfer from BH4 to the
oxygen complex of NOS. Working under different experimental conditions with BH4 analogs, allowed to determine
consecutive elementary steps. Intermediate species were
analysed by UV/Vis absorbance and epr. Depending on the
conditions, the ultimate intermediates were identified as
ferrous and ferric NO complexes. CO binding studies under pressure revealed a kinetic control exerted by BH4.
REFERENCES
1. Renodon-Corniere A., Dijols S., Perollier C., LefevreGroboillot D., Boucher J.L., Attias R., Sari M.A., Stuehr D.J., Mansuy D.: J. Med. Chem.. 45, 944 (2002).
2. Dijols S., Boucher J.L., Lepoivre M., Lefevre-Groboillot D., Moreau M., Frappart Y., Rekka E., Maede A.L.,
Stuehr D.J., Mansuy D.: Biochemistry. 41, 9286 (2002).
3. Moali C., Brollo M., Custot J. Sari M.A., Boucher J.L.,
Stuehr D.J., Mansuy D.: Biochemistry. 39, 8208 (2000).
4. Cox J.D., Cama E., Colleluori D.M., Pethe S. Boucher
J.L., Mansuy D., Ash D.E., Christianson D.W.: Biochemistry. 40, 2689 (2001)
ML12 REGULATION OF ELECTRON
TRANSFER IN NEURONAL
NITRIC-OXIDE SYNTHASE
IKUKO SAGAMI, YUKO SATO, and TORU SHIMIZU
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai
980-8577, Japan, [email protected]
NOS consists of two functional domains: one is an
amino-terminal oxygenase domain with a cytochrome-P450
(P450) like heme active site, the other is a carboxy-terminal
reductase domain that contains the FAD, FMN, and NADPH
binding sites. The two domains are connected by a calmodulin (CaM) binding site. CaM binding activates the interdomain electron transfers required for NO formation as well as
S39
intradomain electron transfers in the reductase domain. Analysis of heterodimers consisting of one full-length nNOS subunit and one oxygenase-domain subunit support an inter-subunit electron-transfer mechanism for the wild-type nNOS, in
that electrons for catalysis transfer in a Ca2+/CaM dependent
way from the reductase domain of one subunit to the heme of
the other subunit, as proposed for iNOS. CaM-dependent regulation of nNOS is also controlled by an autoinhibitory domain identified within the FMN binding subdomain. An autoinhibitory domain deletion mutant (delta40) showed the activity of the wild type enzyme without Ca2+/CaM and electrons in the mutant transfer from the reductase domain of one
subunit to both of the oxygenase domains in a Ca2+/CaM independent way, indicating that the electrons can be transferred via both inter-subunit and intra-subunit mechanisms. However, NO formation activity was exclusively linked to intersubunit electron transfer and was observed only in the presence of Ca2+/CaM.
We also studied NO formation activity in reconstituted
systems consisting of the isolated oxygenase and reductase
domains of nNOS with and without the CaM binding site.
NG-hydroxy-L-Arg (NHA), an intermediate in the physiological NO synthesis reaction, was found to be a viable
substrate. Surprisingly, the NO formation activities with
CaM binding sites on either reductase or oxygenase domains were decreased dramatically on addition of Ca2+/CaM.
Taken together, these results suggest that the mechanism of regulation by CaM is not solely dependent on the
activation of electron transfer to the nNOS hemes, but may
involve additional structural factors linked to the catalytic
action of the heme domain.
In addition to Ca2+/CaM-dependent regulation, caveolin is
known to down-regulate nNOS. To elucidate the mechanism
of regulation by caveolin, we examined the effects of a scaffolding domain peptide of caveolin-1 (82-101: CaV1p1) on
the catalytic activities of several nNOS mutants. CaV1 p1 inhibited Ca2+/CaM-dependent but not independent-NO formation activity and the inhibition was partially recovered in the
presence of excess Ca2+/CaM. The results also indicate that (1)
CaV1p1 can interact with the isolated reductase domain itself,
(2) the presence of CaM is not essential for CaV1p1 inhibition, (3) the autoinhibitory domains of nNOS assist the inhibition of interdomain electron transfer from the reductase domain to the oxygenase domain by CaV1p1.
ML13 STRUCTURAL AND
FUNCTIONAL EVIDENCE
FOR THE EVOLUTION OF
PROKARYOTIC NO SYNTHASES
FROM AN ANCESTRAL
CYTOCHROME P450
C. S. RAMAN
Structural Biology Research Center, Dept. of Biochemistry
and Molecular Biology, University of Texas – Medical
School, Houston, TX , U.S.A. [email protected]
This talk will utilize nitric oxide synthase (NOS) as
a model system to address the question of how novel functions evolve from preexisting protein templates. In mammals NOS is a self-sufficient enzyme in which a heme-thiolate containing oxygenase domain is fused to a di-flavin
reductase domain. NOS catalyzes the five-electron oxidation of L-arginine to NO and L-citrulline. The cofactor tetrahydrobiopterin (H4B) and a high-affinity protein-protein
interaction involving calmodulin (CaM) are a sine qua non
for this reaction to occur. When we discovered NOS-like
proteins in human pathogens, Bacillus anthracis and Staphylococcus aureus, we were surprised to find the catalytic
heme domain in isolation. Although catalytically self-sufficient P450s like BM3 are present in these organisms, a reductase domain is not attached to the C-terminus of the
NOS-like protein. In addition, bacteria that encode a NOSlike protein do not have the capacity to biosynthesize
H4B or CaM. Given these observations, we asked: (a) do
prokaryotic NOS-like proteins function by generating NO?
and (b) what reaction(s) do they catalyze? By combining
high-resolution X-ray crystallography, molecular biology,
proteomics, chemical genetics, and spectroscopy we have
determined that NOS-like proteins have novel enzymatic
activities that share similarities with co-factor independent
ML14 BIOINFORMATIC INSIGHT
INTO THE UNITY AND
DIVERSITY OF CYTOCHROMES
P450
REFERENCES
1. Daff, S. N., Sagami, I., Shimizu, T.: J. Biol. Chem. 274,
30589 (1999)
2. Sagami, I., Daff, S. N., Shimizu, T.: J. Biol. Chem. 276,
30036 (2001).
3. Rozhkova, E., Fujimoto, N., Sagami I., Daff S., Shimizu T.: J. Biol. Chem. 277, 16888 (2002).
4. Yuko Sato, Ikuko Sagami, and Toru Shimizu (submitted
for publication (2003).
ALEXANDER ARCHAKOV, ANDREY LISITSA,
SEMEN GUSEV, and YULIA MIROSHNICHENKO
Institute of Biomedical Chemistry, Pogodinskaya str. 10,
Moscow, 119121, Russia, [email protected]
During the past few decades the cytochromes P450
(CYPs) were the subject of extensive research owing to the
ability of these enzymes to serve as drug targets and to
their active participation in the drug metabolism. Mean-
S40
Table 1.
Model
quality
while other functions and varieties of cytochromes P450
were discovered and currently this superfamily comprises
over 2000 different protein species. In the present study the
protein sequences of cytochromes P450 were submitted to
the computer analysis for elucidation of the structural basics of their pronounced functional diversity. The basic local alignment search tool (BLAST) was used to demonstrate that CYP protein sequences share a certain general
similarity; at the same time, it was shown that the CYP superfamily may be split into a number of groups of intimately related proteins. These groups, the families, were revealed by means of cluster analysis which demonstrated
strong hierarchy among the animal, bacterial and fungal
P450s, and the lack of such hierarchy for the plant enzymes.
Multiple alignment and consensus sequence analysis
were the approaches taken to find out which structural peculiarities of P450s are responsible for the deviations from
the random picture. Proteins within each family were aligned and collapsed to the corresponding consensus sequences, the alignment of which produced the consensus for the
whole superfamily.
By the statistical methods it has been shown, that the
distribution of the conserved positions along family consensus sequences is not even. In particular the conserved
sites tend to make the compact clusters. Due to their nonrandom character it can be expected, that these clusters
constitute the structural-functional motifs.
The superfamily consensus yielded a number of unity
motifs (which are common to many P450 species, and
which are mostly related to the heme-fixing assembly);
whereas the cross-family comparison of consensus sequences enabled to retrieve some of the diversity motifs. Three
consensus sequences (for the CYP51 and CYP2 families
and for the superfamily) were compared to line up the unity
and diversity motifs with the appropriate X-ray data.
good
average
satisfactory
average
satisfactory
good
average
satisfactory
good
average
satisfactory
good
satisfactory
good
average
satisfactory
satisfactory
ML16 GENERAL TRENDS IN 3D
MODELLING OF
CYTOCHROMES P450
satisfactory
ALEXIS IVANOV, VLADLEN SKVORTSOV,
ANDREY LISITSA, and ALEXANDER ARCHAKOV
satisfactory
Moscow, 119121, Russia, [email protected]
average
satisfactory
Currently >1000 P450s sequences are known and only
12 crystal structures (~ 1%) are available from PDB. Apparent deficiency of known 3D structures forces researchers to use the computer modelling. The main method suitable for creation of P450 models is homology modelling
based on sequence and structural similarity between model
and homologues with known 3D structures (templates).
However this approach meets significant difficulties stipu-
average
satisfactory
-
S41
(Template)
Cytochromes P450
(CYP2C5)
2B11-2B12; 2B16; 2B19; 2C1-2C4; 2C6-2C20;
2C22-2C30; 2E1-2E2; 2F4; 2H1-2H2
2A1-2A11; 2A13-2A16; 2B1-2B6; 2B9-2B10;
2B13; 2B15; 2C31-2C32; 2C34-2C36; 2C42;
2D1-2D6; 2D9-2D11; 2D14; 2D16-2D18; 2D25;
2E1; 2F1-2F3; 2G1; 2J1-2J6; 2K4
1A1-1A7; 1B1; 2B7; 2C33; 2L1; 14A1-14A3;
17A1; 18A1; 21A1-21A3; 21B1; 23A1; 33A1;
33B1; 33E1-33E2; 34A5; 34A10; 35C1; 36A1;
71A20; 71D7; 76C2; 76C4; 98A1
(CYP51A)
51A from plants
51A from mammals and yeast
(CYP55A1)
55A1-55A3
105A1; 105A3; 105B1; 105D1
105A2; 105C1; 105E1; 107A1; 107B1; 107C1;
107D1; 107E1; 107G1; 107H1; 107J1; 109B1; 112A;
141A
(CYP101A)
101A
127A
107E1
(CYP102A)
102A; 102A3
3A2; 3A27; 13A6; 72A1; 72A14; 72C1
(CYP107A)
107A01
105E1; 107B1; 107C1; 107G1; 107H1; 107J1
55A1-55A2; 105A1; 105A3; 105B1; 105C1; 105D01;
107D1; 107E1; 107F1; 109A; 109B1; 112A; 112A2;
113A1; 113B1; 114A; 121A; 121A01; 123A; 126A;
130A; 140A; 146A; 154C1
(CYP108A)
107G1; 111A; 124A; 125A; 126A; 130A; 142A
(CYP119A)
106A1-106A2; 107B1; 107H1; 107J1; 109A; 109B1;
113B1; 124A; 142A
(CYP121A1)
55A01; 105A1; 105B1; 105C1; 105D1; 105E1;
107C1; 107D1; 107E1; 107F1; 107G1; 107H1; 112A
(CYP154C1)
107B1
55A3; 105A1; 105D1; 107A1; 107C1; 107D1;
107E1; 107G1; 107H1; 107J1; 111A; 112A2; 123A;
129A; 141A
(CYP165B)
105A; 105E1; 146A
55A1-55A3; 105A; 105A1; 105A3; 105B1; 105C1;
105D1; 107A1; 107B1; 107D1; 107E1; 107G1; 112A;
112A2; 122A1; 140A; 141A
(CYP175A1)
-
lated by low identity of model and template sequences.
CASP1 experiments have shown that the accuracy of homology modelling depends on sequence identity. The model is almost good if identity > 40%. In case of P450s - all
12 known templates belong to different families. At the
same time the classification of P450s is founded on sequences similarity and the mean value of identity for families’ demarcation is 35-40%. Thus some common conclusions can be done: (i) each known structure of P450 can be
used as template only for modelling of P450s from the
same family as template; (ii) mismatching between 1D and
3D alignments of templates does not allow to utilize full
set of known templates for comparative homology modelling; (iii) any attempts to model P450s from other than
template’s family will give the models with considerably
low accuracy especially in the areas of surface loops. In
case of rather low identity a special procedure must be
done to determine the statistical significance of the alignment - comparing the actual alignment score with the average alignment score of random sequences derived from
the original ones. The universal criterion for quality assessment of probable models is alignment significance
score2 (S). We calculated the values of significance score
for all known P450s sequences on the basis of 12 available
templates. The obtained results are presented in Table 1
and can be considered as the prognosis on current state for
P450s modelling. The quality of probable models was estimated by S values: good (50-80), average (30-50), satisfactory (20-30). These prospects of P450s modelling are
increased practically with each new 3D structure. As the
announced structures of human CYP2C9 and CYP3A4 solved by Astex3 were not published in PDB and thus not available for P450 community, the best template for modelling
of several eukariotic P450s nowadays is CYP2C5 (identity
up to 40-60%) with CYP2 family members.
Designed model requires deep optimization by long
time molecular dynamics simulation and energy minimization. However, there is no uniform optimal protocol for
these procedures. Moreover, the problem, what model optimized with or without molecular dynamics simulation is
more adequate to natural protein, is still discussed. So, in
any case all results of molecular modelling must be validated (by structural, topological, molecular dynamics, statistic and experiment verification).
This work was supported in part by Regional Public
Foundation for Support of Domestic Medicine (Russia).
We thank Tripos GmbH (Munich, Germany) for scientific
and technical support.
ML17 CYTOCHROME P450’S FRIENDS
AND FOES: COMPLEX
RELATIONSHIPS UNDER
COMPUTATIONAL ANALYSIS
TIMM ESSIGKEa, PETER WINNb,
MATTHIAS ULLMANNc, AMIT BANERJEEd, and
REBECCA C. WADEA,B
a
European Media Laboratory, Schloss-Wolfsbrunnenweg
31c, 69118 Heidelberg, Germany; bEuropean Molecular
Biology Laboratory, Meyerhofstr. 1, 69012 Heidelberg,
Germany; cIWR - Biocomputing, University Heidelberg,
Im Neuenheimer Feld 368, 69120 Heidelberg, Germany;
d
Institute of Environmental Health Sciences, Wayne State
University, 2727 Second Avenue, Detroit, MI 48201, USA.
[email protected]
Cytochromes P450 are dependent on other proteins for
their in vivo function. Redox proteins act as helper proteins providing electrons for the reaction catalysed by cytochrome P450s. On the other hand, proteasome-dependent
degradation following ubiquitination at specific domains
has been observed for cytochrome P450s1,2. Both types of
P450-protein interaction may influence and be influenced
by substrate binding and turnover as well as post-translational modifications such as phosphorylation. We are applying a range of computational approaches to gain insights
into the determinants of these inter-related contributions to
cytochrome P450 function and xenobiotic regulated turnover. In this presentation, insights from comparative modeling of mammalian cytochrome P450s into binding recognition determinants will first be discussed. Then results of
a combined quantum-mechanical/continuum electrostatics
procedure that we have developed for computing absolute
redox potentials will be shown for two-iron-sulfur cluster
ferredoxins related to those involved in electron transfer
for class I cytochromes P450. Redox potentials can be
computed with good accuracy and the calculations provide
insights into the determinants of the redox potential value
and should aid the design of mutant proteins with altered
redox properties.
REFERENCES
1. Banerjee A, Kocarek, T.A., Novak, R.F. Drug. Metab.
Dispos. 28, 118 (2000).
2. Banerjee A, Wade, R.C. J. Molec. Recogn. 15, 3 (2002).
REFERENCES
1. CASP experiment (http://predictioncenter.llnl.gov).
2. Barton G.J.: Methods in Enzymology, 183, 403 (1990).
3. Astex (http://www.astex-technology.co.uk/one/29_40.html).
S42
posed formalism for describing each P450 through its
structure (i.e. protein sequence) and function (substrate, reaction, inhibitor and other) we describe the rules used to
populate this data fields. These rules appear as a result of
incorporation of the expert’s opinion to the bioinformatic
algorithms and thus we refer the developed system as
a knowledgebase.
ML18 NEW INSIGHTS INTO
MICROBIAL CYP
BIODIVERSITY
STEVEN KELLY, DIANE KELLY, COLIN JACKSON,
ANDREW WARRILOW, TOVE SKAUG,
JOSIE PARKER, and DAVID LAMB
Materials and Methods
The data model for the knowledgebase was proposed
earlier1 in the frameworks of Cytochrome P450 Database.
This Web-based application provides the input and display
for the nomenclature, structure and function of cytochromes P450. To the end of 2001 there were 1200 different enzymatic species in the database.
Wolfson Laboratory of P450 Biodiversity, University of
Wales Aberystwyth, Aberystwyth, Wales, UK.
The post-genomic evaluation of microbes has revealed
unexpected diversity with some possessing a CYP complement even exceeding the numbers found in animals, such
as in the higher basidiomycete fungus Phanerochaete chrysosporium. Most fungi and protists contain CYPs but the
simple eukaryotes Gamblia lamblia and the malarial parasite Plasmodium falciparum did not and are presumably
therefore sterol auxotrophs as at least CYP51 is essential
for sterol synthesis.
Many bacterial genomes have now been sequenced and
an absence of CYP genes is common in the proteobacteria
and archaebacteria where about two-thirds of species fall
into this category. CYP51 has been found in gram positive
and gram negative bacteria and current thinking on the origin will be discussed together with novel CYP forms and
classes recently revealed. The actinomycetes have revealed
unexpected diversity with 1% of all genes being CYPs in
Mycobacterium smegmatis and Streptomyces avermitilis
containing 33 CYP genes involved in many cases in aspects of secondary metabolism. Functional genomic studies on the superfamily in microbes began with yeast
CYP61 and will assist traditional approaches to revealing
the roles of these genes and proteins.
Updating the structural data.
The cross-database comparison has shown that there is
a definite threshold for the results of local alignment searches for new P450s in the global database. These were:
10-4 for the expectation value and 70 for the score. Guided
by these figures we mined the global databases making
each of the available P450s a query in a turn. Best hits were
submitted to global alignment to declare whether the hit is
identical to the existing entry, or it is a fragment, allelic variant, or homologue, which joins the existing (sub)family.
The latter case required the cluster analysis to confirm the
classification2.
Obtaining the facts related to P450 functioning.
The sample of all P450-related PubMed abstracts
(42 000 texts) was compared to the training set of the abstracts that describe the interaction between a P450 form
and a chemical substance. From the comparison we have
captured the words, whose frequency of occurrence assists
in distinguishing the topic of the abstracts. Basing on this
data and on the data from existing database the vocabularies of special terms were produced under the expert-control (e.g. vocabulary of systematic names of P450, of species names, names of low-molecular weight compounds,
reactions, and etc.). Besides, the abstracts’ sentences,
which provide the fact of interaction (or non-intercation)
between P450 and a ligand, were aligned to produce the
stable linguistic constructs - the patterns. Vocabularies and
patterns were used to develop the core of automatic extraction of information from the P450-related texts. The approach has generated the questionnaires for the experts, and
upon the expert-provided correction the data entered the
database.
ML19 BALANCE SHEET FOR
CYTOCHROME P450
KNOWLEDGEBASE:
2000 PROTEIN SEQUENCES,
10000 CHEMICAL SUBSTANCES
ANDREY LISITSA, ALEXANDER ARCHAKOV,
YULIA MIROSHNICHENKO, and
ELENA PONOMARENKO.
Results
We have downloaded more then 500 new P450 sequences to the database. The current distribution of sequences
among families and kingdoms is reported. Peculiarities of
P450-oriented BLAST searches are discussed.
From the natural language processing we have
obtained the vocabulary of over 10000 names of chemical
Moscow, 119121, Russia, [email protected].
Introduction
The overall growth of the amount of information on cytochromes P450 requires the automatic means for parsing
and annotation of the data. Relying on the previously pro-
S43
substances that were mentioned in relation to cytochromes
P450. It was shown that the patterns are an effective tool to
unravel the special terms from the biomedical text, especially when the results undergo extra filtering. The filtering
included the comparison of the compound’s name to MeSH
(Medical Subject Headings) collection of terms. In 75% of
cases the expert confirmed that the automatically selected
abstract contains the information related to the interaction
between the P450 from and drug/chemical substance. Parsing of the abstract demanded the correction in 68% of cases, but even in this case the correction required the
„yes/no“ answer and thus the efficiency of data input was
high. The number of facts (P450 name - ligand) stored during the 2 months of work exceeded twice the manual input
of the last five years. Currently the knowledgeable incorporates in total ~1900 facts, each in the relation to the corresponding MedLine record.
Conclusions
P450-related research provides a field, where the automatic means of data mining and annotation can be extremely effective. Their further development and application
should enable the accumulation of enough facts, and these
facts, combined with the standard QSAR and 3D bioinformatics will enable the real steering of the experimental
work.
Acknowledgements
The work is supported in part by the contracts with
Russian Ministry of Industry, Science and Technology and
Janssen Research Foundation (Belgium).
REFERENCES
1. Archakov A.I., Lisitsa A.V., Zgoda V.G., Ivanova M.S.,
Koymans L.: J Mol Model 4, 234 (1998).
2. Lisitsa A.V., Gusev S.A., Karuzina I.I., Archakov A.I.,
Koymans L.: SAR QSAR Environ Res 12, 359 (2001).
S44
2
LECTURES – TUESDAY – TL
TL01
RATE-LIMITING STEPS
IN CYTOCHROME P450
REACTIONS
Invited Lectures – TL
P450s 2E1 and 2A6
Work on the oxidation of ethanol and acetaldehyde by
P450 2E showed deuterium isotope effects on Km but not
kcat, due to the burst kinetics in these reactions. 6, 7 In a simplified reaction with a slow kinetic step following product
formation, the expressions for kcat and Km are complex and
Km is a function of k3, the C-H bond-breaking step.
Studies on the oxidation of N,N-dimethylnitrosamine
show isotope effects on Km (DK) of ~3 with human P450s 2E1
and 2A6. However, isotope effects approach unity for both
enzymes in the oxidation of N,N-diethylnitrosamine. Studies with asymmetric N-methyl, N-ethylnitrosamine are in
progress to understand the phenomenon, as well as more
studies on the oxidation of HCHO and CH3CHO.7
F. PETER GUENGERICH, M. WADE CALCUTT,
JOEL A. KRAUSER, GROVER P. MILLER,
MARTHA V. MARTIN, IMAD H. HANNA, and
HIDEAKI SATO
Department of Biochemistry and Center in Molecular
Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, U.S.A. [email protected]
P450 3A4
Testosterone 6β-hydroxylation is one of the most used
assays of P450 3A4 activity and has one of the highest rates. The 6-d2 substrate has been prepared and initial studies
show DV ~ 5. Although much of the modeling work on
P450 3A4 has assumed that the enzyme has little specific
bonding ability and does oxidations based largely on ease
of bond breaking,8 this is one of the few experiments to test
this hypothesis. Whether isotope effects are seen with more
slowly oxidized substrates is yet unknown. Current investigations are also directed to examination of the kinetic
contribution to the cooperativity seen in some P450 3A4
reactions, including testosterone 6β-hydroxylation.9,10
Cytochrome P450 (P450) enzymes use a complex catalytic cycle, usually described with a minimum of 9 steps
even without including any conformational changes. A general perception often imparted in the literature is that most
P450 reactions are limited by the rate of transfer of either
the first or second electron to the P450 heme, and another
is that catalytic activity towards a substrate is simply the
result of good docking in an active site. We have examined
intermolecular kinetic hydrogen isotopes on kcat (DV) and
Km (DK) as a means of investigating the rate-limiting aspect
of C-H bond cleavage in P450 reactions. Both DV and
D
(V/K), the isotope effect on the ratio kcat/Km, are considered in interpretations of P450 mechanisms.1
Summary
High intermolecular kinetic deuterium isotope effects
have been found in several reactions catalyzed by human
P450 enzymes. The results suggest that C-H bond breaking
can be partially rate-limiting but that several catalytic steps
contribute to the observed kcat values.5, 11 The results also indicate that Km is still a complex and poorly understood parameter in most P450 reactions.
P450 2D6
D
V and D(V/K) for the O-demethylation of 3- and 4-methoxyphenethylamine were in the range of 3-4.2 This particular case is complicated by an apparent substrate inhibition at high concentrations; evidence has been presented
that this inhibition can be explained by the oxidation of
substrate to an inhibitory nitroso product.2 The mutation
E216Q decreased kcat/Km 400-fold but did not change
D
(V/K).3
REFERENCES
1. Northrop D.B.: Methods Enzymol. 87, 607 (1982).
2. Guengerich F.P., Miller G.P., Hanna I.H., Sato H.,
Martin M.V.: J. Biol. Chem. 277, 33711 (2002).
3. Guengerich F.P., Hanna I.H., Martin M.V., Gillam
E.M.J.: Biochemistry 42, 1245 (2003).
4. Yun C.-H., Miller G.P., Guengerich F.P.: Biochemistry
39, 11319 (2000).
5. Yun C.-H., Miller G.P., Guengerich F.P.: Biochemistry
40, 4521 (2001).
6. Bell, L.C., Guengerich F.P.: J. Biol. Chem. 272, 29643
(1997).
7. Bell-Parikh L.C., Guengerich F.P.: J. Biol. Chem. 274,
23833 (1999).
8. Smith D.A., Jones B.C.: Biochem. Pharmacol. 44, 2089
(1992).
9. Harlow G.R., Halpert J.R.: Proc. Natl. Acad. Sci. USA
95, 6636 (1998).
P450 1A2
We previously reported DV and D(V/K) values of ~ 3 for
the O-dealkylation of phenacetin and >10 for acetol formation with human P450 1A2.4, 5 The basis of the different
isotope effects for the two different reactions with the same
substrate is still under investigation.
Further work with (rabbit) P450 1A2 shows high DV values for the O-dealkylation of other 4-substituted anisoles,
in the range of 7-9.
The values for the intermolecular kinetic isotope effects
are still less than the intramolecular isotope effects measured for the demethylation reactions and are consistent with
partially-limiting C-H breaking. However, kcat still appears
to be less than the rate of bond breaking, with a contribution of a step late in the oxygen activation process with
both P450s 2D6 and 1A2.2, 5
S45
10.Hosea N.A., Miller G.P., Guengerich F.P.: Biochemistry
39, 5929 (2000).
11. Guengerich F.P.: Chem. Res. Toxicol. 14, 611 (2001).
TL02
7. Shaik, S., de Visser, S. P., Ogliaro, F., Schwarz, H.,
Schröder, D.: Curr. Opinion Chem. Biol., 6, 556(2002).
8. Newcomb, M., Toy, P. H.: Acc. Chem. Res., 33, 449
(2000).
9. Auclair, K., Moënne-Loccoz, P., Ortiz de Montellano,
P. R.: J. Am. Chem. Soc., 123, 4877 (2001).
10. De Visser, S. P., Ogliaro, F., Sharma, P. K., Shaik,
S. Angew. Chem. Int. Ed., 41, 1947 (2002).
11. De Visser, S. P., Ogliaro, F., Sharma, P. K., Shaik, S.:
J. Am. Chem. Soc., 124, 11809 (2002).
THE PROTEIN, THE ACTIVE
SPECIES AND THE
CHAMELEON: REACTIVITY
PATTERNS OF OXYGEN
TRANSFER BY CYTOCHROME
P450
TL03
SASON SHAIK
Department of Organic Chemistry and The Lise Meitner
Minerva Center for Computational Quantum Chemistry,
The Hebrew University, 919104, Jerusalem,
[email protected]
P450’S PECCANT PROTON
PATHWAYS: PERICLESIAN
CONTROL OF ACTIVE OXYGEN
STEPHEN SLIGARa,b, THOMAS MAKRISb,
ILIA DENISOVa,b, and ILME SCHLICHTINGc
a
Departments of Biochemistry, Chemistry, the College of
Medicine; bCenter for Biophysics, University of Illinois,
505 S. Goodwin Avenue, Urbana, IL 61801 USA; cMax
Planck Institute for Molecular Physiology, Department of
Molecular Biochemistry, Otto-Hahn-Straße 11, D-44227
Dortmund, Germany.
Theoretical results on Compound I (Cpd I) and its reactivity patterns in hydroxylation and epoxidation will be
reviewed.1-9 I shall begin by showing that Cpd I behaves
like a chameleon species that adopts its geometry and electronic structure to the protein environment in which it is
accommodated.1-4 I shall then proceed to show that the mechanisms of oxygen transfer by Cpd I involve two-state reactivity (TSR), and will argue that this mechanism leads to
a resolution of the controversy5-7 regarding the rebound mechanism of alkane hydroxylation.8,9 Subsequently, I will
discuss recent model calculations of C-H hydroxylation
and C=C epoxidation pathways, which show that not only
Cpd I is a chameleon state, it is also a chameleon oxidant
that changes its reactivity and selectivity patterns under the
influence of hydrogen bonding and polarization effects,
which mimic the protein environment.10.11 I will argue that
using a chameleon species in many different protein environments is one of the ways by which the enzyme modulates its selectivity. The Chameleon oxidant can serve as
a new paradigm in enzyme catalysis.
The desire for a detailed understanding of the chemistry
by which the cytochromes P450 catalyze substrate oxidation events has been paramount in the field since the discovery of these ubiquitous heme-thiolate oxygenases
a half-century ago. Progress towards this goal has often
been measured by the identification of the critical intermediate states of heme- iron and the staged reduction steps of
atmospheric dioxygen during the catalytic cycle. Identification of the ferrous-O2 complex in the early 1970s focused attention on the features of the cytochromes P450
which were distinct from that of the globins which, in their
normal function, reversibly bound dioxygen without „activation“ to a state chemically competent to transfer an oxygen atom to a recalcitrant carbon center. Yet various spectroscopic tools utilized in the 1970s and 1980s showed the
oxygen complexes in these systems to have many shared
features, suggesting that the critical differences in chemical
reactivity were due to control by protein residues in the active site. A great breakthrough was provided by Poulos and
colleagues in the early 1980s with the crystal structure of
the ferric resting state of P450cam CYP101. The gene sequence of CYP101 and its heterologous expression opened
the Pandora’s box of mutagenesis to build on the road map
provided by the Poulos structure. A wealth of modern bioorganic and bioinorganic chemical approaches with native
and mutant enzymes followed and provided tantalizing
hints as to the subtle control of electrons and protons in the
processes of oxygen activation and substrate functionalization. However, despite these great advances, the close of
the 20th century saw no new reactive intermediates of the
REFERENCES
1. Ogliaro, F., Cohen, S., Filatov, M., Harris, N., Shaik, S.:
Angew. Chem. Int. Ed., 39, 3851 (2000).
2. Ogliaro, F., Cohen, S., de Visser, S. P. , Shaik, S.: J. Am.
Chem. Soc., 122, 12892 (2000).
3. Ogliaro, F., de Visser, S. P., Cohen, S., Kaneti, J., Shaik,
S.: ChemBioChem., 2, 848 (2001).
4. Schöneboom, J. C., Lin, H., Reuter, N., Thiel, W.,
Cohen, S., Ogliaro, F., Shaik, S.: J. Am. Chem. Soc.,
124, 8142 (2002).
5. Ogliaro, F., Harris, N., Cohen, S., Filatov, M., de Visser,
S. P., Shaik, S. : J. Am. Chem. Soc., 122, 8977 (2000).
6. De Visser, S. P., Ogliaro, F., Harris, N., Shaik, S.:
J. Am. Chem. Soc., 123, 3037 (2001).
S46
P450 reaction cycle cleanly defined by structural or spectroscopic tools.
Beginning in the new millennium, however, x-ray
structure of the ferrous dioxygen bound state of CYP101
was determined. This structure revealed subtle but critical
conformational changes of P450 upon dioxygen binding
and pointed to a dynamic active site where water and side
chain motion contribute to the critical hydrogen bonding
network responsible for delivery of protons for productive
oxygenation and/or uncoupling events. More recently, the
use of cryoradiology and thermal annealing together with
state-of-the art resonance and optical spectroscopies defined two new sequential intermediates of P450 catalysis,
the peroxo- and hydroperoxo- states. In my presentation,
I will describe our current understanding of the intermediates and proton / electron transfer events that describe the
monoxygenase process. I will also describe very recent unpublished results. For instance, with further structural analysis, spectroscopic investigations and additional active
site mutations in the cytochrome P450s we can, for the first
time, separate the defining processes of proton delivery
which lead to (1) the interconversion of peroxo anion and
hydroperoxo states, (2) the protonation leading to dioxygen
bond scission and (3) the very different pathway open in
peroxide uncoupling. Similar intermediate states are observed in other heme-oxygen systems, which leads to a unifying picture of the commonality of the peroxo-state and the
linkage of protein dynamics to control water and side-chain
positioning to define the „proton wires“ of dioxygen activation by the P450 monoxygenases.
Addition of a second electron yields a peroxo-ferric intermediate, protonation of which generates a hydroperoxoferric state; lose of water then forms an oxo-ferryl porphyrin
radical (Compound I). Although the latter is thought to be
the ultimate oxidant, the peroxo- and hydroperoxoferric
species have been proposed as secondary oxidants. The
T252A mutant of P450-CAM was first prepared by Sligar
and Ishimura and their co-workers. Because this mutant
does not hydroxylate camphor, it must not form P450
Compound I. However, it still accepts electrons from
NADH to give hydrogen peroxide, presumably via the peroxo- and hydroperoxoferric intermediates. Thus,
T252A P450-CAM is the ideal mutant to test whether one
and/or the other of these two species are capable of O-atom
transfer. We have prepared a series of camphor analogues
that contain reactive functional groups that are the site of
O-atom transfer in order to investigate the mechanism of
olefin epoxidation, ether O-dealkylation, amine N-dealkylation, thioether sulfoxidation and N-hydroxyimine denitrosation. With 5-methylenylcamphor, for example, we find
that the T252A mutant is enzymatically active in olefin
epoxidation, producing epoxide at 20% of the wild type
rate. This clearly demonstrates that a second active oxidant
is formed in the P450 reaction cycle. Results of our studies
of oxygen activation by P450 with these model substrates,
with emphasis on the involvement of a second oxidant in
O-atom transfer, will be presented.
Funding from NIH (GM 26730). We thank Steve Sligar
for providing the over expression system for T252A P450CAM.
TL04
TL05
OXYGEN ACTIVATION BY
CYTOCHROME P450 AND
NITRIC OXIDE SYNTHASE.
MECHANISTIC EVIDENCE
FROM STUDIES WITH
P450-CAM AS A MODEL
SYSTEM
MOLECULAR MECHANISM
OF P450CAM CATALYZED
OXYGENATION REACTION
REGULATED BY THE
ASSOCIATION WITH
PUTIDAREDOXIN
ISAO MORISHIMA
SHENGXI JIN, MARY C. LAMCZYK,
HEATHER L. VOEGTLE, THOMAS A. BRYSON, and
JOHN H. DAWSON
Department of Molecular Engineering, Graduate School of
Engineering, Kyoto University, Kyoto 606-8501, Japan,
[email protected]
Department of Chemistry and Biochemistry, University of
South Carolina, Columbia, SC 29208 USA
In addition to being an electron shuttle to P450cam, Putidaredoxin (Pdx ) is a conformational effector of P450cam.
Low potential iron sulfur protein such as spinach ferredoxin and bovine adrenodoxin can transfer a first electron to
P450cam but not a second electron, resulting in a very slow
turnover of the P450cam monooxygenase reaction. This
conformational change of P450cam induced by the binding
of Pdx has remained to be solved. In order to characterize
the Pdx-induced conformational changes in P450cam, we
applied proton NMR to the P450cam-Pdx complex, and
Cytochrome P450 is a versatile heme-containing oxygenase that transfers oxygen atoms from dioxygen to
a wide range of organic substrates. Nitric oxide synthase is
also an oxygenase; it first hydroxylates arginine to an Nhydroxyimine intermediate and then, in a second O-atom
transfer step, forms citrulline and NO. Hydroxylation involves substrate binding to the ferric enzyme followed by
reduction and oxygen binding to give the oxyferrous state.
S47
also utilized the L358P mutant of P450cam as a model for
the Pdx-bound P450cam, In the ring current shifted proton
NMR spectra of carbon monoxy P450cam, the signals from
the heme iron-bound Cys 357 protons, Thr 252 protons in
P450cam, and 5-exo and 9-methyl protons of d-camphor
were assigned. The Pdx-induced shifts for these proton signals suggest that the axial Cys moves closer to the heme
iron, and that d-camphor moves toward the heme iron by
0.15~0.5 ■ . To gain further insight into the Pdx-induced
structural changes in P450cam, we utilized L358P mutant
as a model for Pdx bound P450cam, since the proton NMR
spectrum of the L358P mutant was quite similar to that of
Pdx bound P450cam. The X-ray crystallographic study of
the CO form of L358P mutant showed that the heme plane
tilts and Arg112, the site of binding with Pdx, is substantially dislocated in the mutant as compared with the wild
type P450cam, in agreement with the NMR results. Moreover, the L358P mutant exhibited an enhanced association
property with Pdx as compared with the wild type
P450cam. This prompted us to examine the reactivities of
oxy-L358P with the non-physiological electron donors
such as dithionite and ascorbic acid. We observed enhancement of the cross reactivity with these non-physiological
donors. Taken together, Pdx-induced structural changes in
P450cam facilitate the monooxygenation reaction.
TL06
ently from their correspending heme thiolate complexes,
e.g. see 5 to 3, Fig. 2.
By comparison of 3 and 5 it is obvious that by exchanging S- for SO3- a very significant anodic shift of
285mV to E0 = -175mV can be observed which, within
experimental error, coresponds exactly to the value of the
high-spin E.S complex of cytochrome P450cam. The complexes 3 and 4 not only resemble the native cofactor regarding electrochemistry but also with respct to reactivity. In this context regioselective epoxidation and halogenations of various substrates were catalyzed by 3 +
PhIO and 4 + NBu4OCl, respectively. Turnover approached 1800 at 0.1mol% catalyst concentration. The experimental results suggest that the active, oxidizing species
generated in situ from e.g. 4 and PhIO is electronically similar to compound I, this conclusion is supported by DFT
calculations.[6]
DESIGN AND SYNTHESIS OF
NEW P450 ENZYME MIMICS
Fig. 1. First generation P450 enzyme models of the resting state
of P450cam
WOLF-D. WOGGON, TYCHO LEIFELS, and
LAURA SBARAGLI
Department of Chemistry, University of Basel, St. Johanns
Ring 19, CH-4056 Basel, Switzerland,
[email protected]
P450 Enzyme models from our laboratory such as 1 and
2 (Fig 1), carrying aryl thiolate or alkyl thiolate ligands coordinating to Fe(III) displayed Eo ~ -600 and ñ700mV, that
is far more negative than the corresponding values of the
the resting state of P450cam (Eo = -300mV).[1], [2], [3]
Subsequently it was reported that the thiolate ligand in
P450cam is hydrogen-bonded to amino acid residues of the
protein backbone,[4] such that the negative charge at sulfur
is distributed over a larger space segment than originally
believed.[5] Since the electron donating character, i.e. the
„point charge”, at the fifth ligand coordinating to iron is the
decisive factor to the redoxpotential of the heme-thiolate
cofactor we decided to modify the proximal coordination
site of 1 towards a more „diffuse” neagtive charge at/near
sulfur in order to mimic more closely the natural ligand sphere.
We reasoned that such a „diffuse” negative charge
could be created using a SO3- ligand coordinating to iron,
we thus synthesized the cofactor models 3 and 4 conveni-
Fig. 2. P450cam resting state models carrying a SO3- ccordinating
to iron
S48
of the enzyme is still unknown despite decades of efforts
but a large number of studies have been performed in order
to better understand the mode of substrate/inhibitor/effector binding in the active site. These studies are of interest
from a basic enzymology point of view, but also for practical applications. A large number of drug-drug interactions,
leading to toxic or even lethal effects can indeed be explained by mutual inhibition at the CYP3A4 level of two coadministered compounds3.
Recent studies based on UV-Vis spectral titrations4 or
fluorescence measurements5 lead to the assumption that
multiple substrates can bind within the active site, each having access to compound I. Models have been proposed to
explain the behaviour of the different compounds tested
but none of these models is able to predict the behaviour of
a new ligand. This would be indeed of great importance for
the screening of new drug candidates and the evaluation of
their ability to produce drug- drug interactions at the
CYP3A4 level.
We have designed and synthesized new CYP3A4 probes with the aim to rapidly screen the affinity of new compounds versus the CYP3A4 and to use these tools in the investigation of the CYP3A4 active site.
Favourably these compounds should be designed in
such a way that their affinity to CYP3A4 could be measured directly and their displacement from the active site by
potential drugs evaluated easily without performing the
usual inhibition tests on a model substrate. We have therefore designed fluorescent derivatives whose binding to the
CYP heme should be easily followed by direct spectral
measurements such as fluorescence. Furthermore these inhibitors should display a Ki between 1-10 µM, which is
considered to be a cut-off value to eliminate drugs binding
too strongly to CYP 3A4 (i.e. able to
displace the inhibitor from the
CYP3A4 active site). We report here
on the synthesis and the first tests performed with these new probes.
REFERENCES
1.
2.
3.
4.
5.
Stäubli B., PhD Thesis University of Zurich 1989.
Ghirlanda S. L., PhD: Thesis University of Zurich 1994.
Woggon W.-D.: Chimia 55, 366 (2001).
Poulos T. L.: J. Biol. Inorg. Chem. 1, 356 (1996).
Poulos T. L., Finzel B. C., Howard A. J.: Biochemistry
25, 5314 (1986)
6. Lyfels T., Kozuch S., Sbaragli L., Shaik S.,. Woggon W.-D,
in preparation.
TL07
DESIGN AND SYNTHESIS OF
NEW FLUORESCENT PROBES
FOR EVALUATION OF CYP3A4
DRUG-DRUG INTERACTIONS
ANTOINETTE CHOUGNET, DOMINIK MEYER,
MICHAEL MÜLLER, DAVID RICARD, CATHERINE
STOESSEL, and WOLF-D. WOGGON
Institute of Chemistry, University of Basel, St. JohannsRing 19, CH-4056 Basel, Switzerland
[email protected]
Cytochrome P450 3A4 (CYP3A4), the most abundant
isoform in human liver and intestine, oxidizes many endogenous compounds as well as about half of the drugs on the
market1,2. Although this enzyme shows broad substrate selectivity, it still exhibits high regio-and stereoselectivity.
Furthermore the CYP3A4 catalyzed reactions present complex, non-Michaelis-Menten kinetics. The X-ray structure
The synthesis of one of the compounds synthesized as probe for CYP
3A4, the deazatestosterone derivative
6 is depicted in Figure 1. This derivative of testosterone should obviously
bind to the „testosterone site“ and by
ππ-stacking to the heme-thiolate of
CYP 3A4 as indicated in Figure 2.
Compound 6 was used as inhibitor
for the four model reactions generally
used in inhibition tests, i.e. the metabolism of testosterone, midazolam, nifedipine and benzyloxycoumarine.
Fig. 1. Synthesis of compound 6
S49
TL08
Fig. 2. proposed
interaction of compound
6 with the heme-thiolate
cofactor of CYP3A4
OVERVIEW OF CYP GENE
REGULATION WITHIN
A TANGLE OF REGULATORY
NETWORKS
JEAN-MARC PASCUSSIa, SABINE GERBAL-CHALOINa,
LIONEL DROCOURTa, ERIC ASSÉNATa,b,
DOMINIQUE LARREYa,b,
LYDIANE PICHARD-GARCIAa,
MARIE-JOSÉ VILAREMa, and PATRICK MAURELa
a
INSERM U128, 1919 Route de Mende, 34293 Montpellier
France; b Hépato-Gastroentérologie, CHU Saint Eloi,
34295 Montpellier France, [email protected]
These four substrates are known to bind at different ligand
binding sites in the CYP3A4 active site4,6. CYP3A4 selectively expressed in Baculovirus-insect-cells (Gentest-BD
Biosciences) was used in these studies. The IC 50 found with
the four model substrates are all of the same range of magnitude (3, 0.5, 4.5 and 2 µM using testosterone, midazolam,
nifedipine and benzyloxycoumarine, respectively) indicating that the probe compound 6 inhibits with the same potency the metabolism of different substrates, irrespectively
of their binding in the active site.
It is easy to obtain the C-6α epimer of compound 6 by
treating the testosterone derivative 4 under acidic conditions and coupling with the deazaflavine moiety 5 as indicated in figure 1. The IC50 obtained with the 6α-6 are very similar to those obtained with the 6β isomer (2, 7 and
8 µM using midazolam, testosterone and nifedipin as substrates).
These results show that the two testosterone derivatives
epimeric at C-6 may bind in the active site as indicated in
figure 2.
Compound 6 and isomers are therefore good candidates
for achieving a general binding of all sites present in
CYP3A4. This is of great advantage in the evaluation of
new drug candidates as ligands for CYP3A4 and offer new
possibilities for testing their safety in co-medication with
other drugs.
During species evolution, a complex signalling network, consisting of cytochromes P450 (CYPs) and other
xenobiotic metabolizing and transporting systems
(XMTS), and of specific nuclear xenoreceptors such as aryl
hydrocarbon receptor (AhR)1, pregnane X receptor (PXR)2
and constitutive androstane receptor (CAR)3, has been set
up to cope with the risks presented by xenobiotics4. This
network evolved in parallel with other signalling networks
controlling endogenous metabolism. The consequence of
this is that CYPs and XTMS may not be only regulated by
xenobiotics, but also by physiopathological stimuli including glucocorticoids, sterols, biliary salts, pro-inflammatory cytokines and presumably others.
It has been known for many years that the AhR (member of the PAS family) controls CYP1A and other XMTS
gene expression1,4. More recently, two xenoreceptors PXR
(NR1I2) and CAR (NR1I3), activated by xenobiotics and
drugs, have been identifed and characterised as members of
the hormone nuclear receptor superfamily2,3. These receptors control the expression of CYP2 and CYP3A families,
as well as of other XMTS, notably UDP glucuronyl- and
glutathione-S-transferases, MDR1, and MRP2. Studying
these regulations is important from a clinical point of view
because they play a major part in the process of detoxication and AhR- as well as PXR/CAR-dependent enzyme induction is one of the major causes of drug interactions.
Upon activation in response to xenobiotics such as TCDD,
and rifampicin or phenobarbital, AhR and PXR or CAR
transactivate their target genes as heterodimers with ARNT
(AhR nuclear translocator) and RXR (retinoid X receptor),
respectively. Human hepatocyte cultures represent the model of choice for investigating these receptors and their role
on the CYP and XMTS gene induction. Indeed, the species-specificity that prevails in these processes precludes
extrapolations from animals (rodents) to man.
We have developed specific tools (genomic clones, probes, antibodies) for PXR, CAR, glucocorticoid (GR) and
vitamin D receptor (VDR, NR1I1). We investigated their
regulation, functional interactions and role in the inducible
expression of CYP2 and CYP3A genes. Our results showed
that: i) expression of PXR and CAR is a limiting factor in
REFERENCES
1. Guengerich F.P. in: Cytochromes P450 (Ortiz de Montellano P.R.,ed.), pp 473-535, Plenum Press, New York
2. Lu A.Y.H.: Drug Metab. Dispos. 26, 1217 (1998)
3. Lilja J., Kivisto K., Neuvonen P.: Clin Pharmacol Ther,
68, 384 (2000); Lin J.H., Lu A. Y. H.: Pharmacol Rev,
49, 403 (1997); Guengerich F.P.: Ann Rev Pharmacol
Tox, 39, 1 (1999)
4. Hosea N.A., Miller G.P., Guengerich F.P.: Biochemistry
39, 5929 (2000)
5. Dabrowski M.J., Schrag M.L., Wienkers L.C., Atkins
W.M.: J. Amer. Chem. Soc. 124, 11866 (2002)
6. Wang R.W., Newton D.J., Liu N., Atkins W.M., Liu
A.Y.H.: Drug Metab. Dispos. 28, 360 (2000)
S50
the detoxication process5,6; ii) expression and activity of
these receptors is increased by glucocorticoids and decreased by pro-inflammatory cytokines (IL-1 and IL-6)5-7; iii)
GR indirectly controls detoxication via the signal transmission cascade GR-PXR/CAR-CYP2/CYP38; iv) CYP2C9
expression (one of major form present in adult human liver) is under the control of both GR and PXR/CAR, and we
identified and characterised the 5’-response elements involved in this control9; v) VDR is able to bind to PXR- and
CAR-“specific“ response elements on CYP2B6, 2C9 and
3A4, and transactivate these genes10. This last observation
is of critical importance. It shows that there are possible
functional cross-talks between nuclear receptors whose
specificity for their target genes is not as strict as previously thought. On important consequence of this is that xenobiotics and drugs might affect signalling pathways of
endogenous metabolism including notably biliary salt and
Vitamin D homeostasis. The aim of this lecture is to review: i) the recent developments on the mechanisms of induction, including the role of nuclear receptors such as
AhR, PXR and CAR; ii) the possible interferences between
xenobiotic-signalling pathways leading to CYP gene induction and other endogenous signalling pathways.
TL09
MOLECULAR MECHANISMS OF
TRANSACTIVATION OF AH
RECEPTOR IN THE TARGET
GENE EXPRESSION
Y. FUJII-KURIYAMAa, J. MIMURAa,
H. MOTOHASHIa, M. YAMAMOTOa, F. OHTAKEb,
S. KATOb., K. NUMAYAMA-TSURUTAc,
K. SOGAWAc, T. BABAd, and K. MOROHASHId
a
Center for Tsukuba Advanced Research Alliance and Institute for Basic Medical Sciences, University of Tsukuba,
Tsukuba 305-8577, [email protected] bInstitute of
Molecular and Cellular Biosciences, The University of Tokyo, Bunkyou-ku Tokyo 113-0032, cGraduate School of Life
Science, Tohoku University, Sendai 980-8578, dNational
Institute for Basic Biology, Nishigonaka 38, Myodaiji,
Okazaki Aichi 444-8585, Japan.
Arylhydrocarbon receptor was first identified as a binding factor to polycyclic aromatic hydrocarbons including
2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene (3MC). The binding affinity of AhR was
found to be extremely high toward TCDD and from genetic studies using mice, it was suggested that the AhR was
involved in the inducible expression of xenobiotic metabolizing enzymes such as CYP1A1, 1A2 and UDP-glucuronosyltransferase. Molecular biological studies on the induction mechanism of the xenobiotic metabolizing enzymes led us to molecular cloning of AhR, revealing that
AhR is a transcription factor with a charcteristic structural
motif of bHLH-PAS which functions in association with
a partner molecule, Arnt to activate the expression of the
target genes in response to added inducers.
Under normal conditions, AhR exists in the cytoplasm
in a complex with HSP90, XAP2 and P23. Upon binding
with a ligand, the AhR complex translocates to the nuclei
where AhR switches its partner molecule from the HSP90
complex to Arnt and then binds the XRE (xenobiotic response element) sequence in the promoter of the target genes to tansactivate their expression.
Although expression of CYP1A2 gene is also induced
by dioxins through AhR and Arnt, its mode of expression
is different from that of CYP1A1 gene whose expression is
a prototype of the genes driven by the XRE sequence.
While CYP1A1 is induced ubiquitously in various tissues
by administration of inducers, both the constitutive and inducible expression of CYP1A2 gene is specifically limited
to the liver. In AhR-KO mice, the constitutive expression
of CYP1A2 gene was still retained, while the expression of
CYP1A1 gene was totally abolished together with the loss
of the inducible expression of CYP1A2 gene.
By DNA transfection experiments using the fusion
gene consisting of the luciferase gene and the CYP1A2
gene upstream promoter sequence up to -9kb, we identified
the inducible enhancer sequence of CYP1A2 gene which is
REFERENCES
1. Hankinson, O.: Annu Rev Pharmacol Toxicol. 35, 307
(1995).
2. Kliewer, S. A., J. T. Moore, L. Wade, J. L. Staudinger,
M. A. Watson, S. A. Jones, D. D. McKee, B. B. Oliver,
T. M. Willson, R. H. Zetterstrom, T. Perlmann, and J.
M. Lehmann:, Cell. 92, 73 (1998).
3. Honkakoski, P., I. Zelko, T. Sueyoshi, and M. Negishi:
Mol Cell Biol. 18, 5652 (1998).
4. Gonzalez, F. J.: Pharmacol Sci. 13, 346 (1992).
5. Pascussi JM, Drocourt L, Fabre JM, Maurel P, Vilarem
MJ.: Mol Pharmacol, 58, 361 (2000).
6. Pascussi JM, Gerbal-Chaloin S, Fabre JM, Maurel P,
Vilarem MJ.: Mol Pharmacol 58, 1441 (2000).
7. Pascussi JM, Gerbal-Chaloin S, Pichard-Garcia L, Daujat M, Fabre JM, Maurel, Vilarem MJ.: Biochem Biophys Res Commun 274, 707 (2000).
8. Pascussi JM, M Busson-Le Coniat, P Maurel, MJ Vilarem: Mol Endocrinol 17, 42 (2003).
9. Gerbal-Chaloin S, Daujat M, Pascussi JM, Pichard-Garcia L, Vilarem MJ, Maurel P.: J Biol Chem 277, 209
(2002).
10.Drocourt L, Ourlin JC, Pascussi, JM Fabre, JM, Maurel
P, Vilarem MJ.: J Biol Chem 277, 25125 (2002).
S51
different from the XRE sequence of CYP1A1 gene. The
enhancer sequence of CYP1A2 gene did not directly bind
with the AhR and Arnt heterodimer, but interacted with it
through factors directly binding to the sequence. Purification with affinity chromatography using CYP1A2 enhancer
sequence and sequence analysis of the binding factor revealed that the binding factors were LBP-1a and 1c.
Transfection of the expression plasmid of LBP-1a or
1c enhanced the reporter gene expression driven by the
CYP1A2 enhancer in response to the added inducer and
this enhanced expression of the reporter gene were further
strengthened by coexpression with AhR and Arnt. GST
pull-down assay showed that LBP-1a and 1c physically interacted with AhR and Arnt. These results demonstrated
that AhR and Arnt activate the expression of CYP1A2 gene
as a coactivator in response to the inducers. The DNA binding site of the AhR/Arnt heterodimer was not necessary
for the coactivator function.
This coactivator-like function of AhR/Arnt heterodimer
was also observed with transactivation of ER (estrogen receptor) target genes such as cFOS and VEGF by interaction
of non-liganded ER with the ligand-activated AhR/Arnt.
The reporter gene consisting of the luciferase structural
gene driven by the ERE (estrogen response element) sequence was activated by 3MC through AhR/Arnt in the presence of ER α or β without E2. In this case, 3MC-activated
AhR/Arnt interacted with ER bound on the ERE sequence
to activate the expression of the reporter genes. The AF-1
region of ER was found to be the site for interaction with
the heterodimer of AhR and Arnt, and the N-terminal DNA
binding site of Arnt was not necessary for the coactivator
function. These results indicate that the coactivator-like
function is another common transcriptional role for AhR
and Arnt.
We will also talk about functional roles of AhR in reproduction of female mice by using AhR knockout mice.
lear receptor CAR (NR1I3) by proinflammatory cytokines
such as interleukin-1β (IL-1β) and lipopolysaccharide
(LPS) in human hepatocytes. CAR is a key transactivator
of CYP2B and CYP3A, among others, upon phenobarbital
administration. We found that IL-1β and LPS decrease
CAR expression and suppress phenobarbital-mediated
CYP2B6 and CYP3A4 induction in human hepatocytes.
Moreover, our data suggest that activation of nuclear factor-kB (NF-kB) is a critical event in LPS- or IL-1β-mediated inhibition of CAR expression through the repression of
ligand-activated glucocorticoid receptor action. Further,
we observed that NFkB p65 interferes with the enhancer
function of the distal glucocorticoid response element of
the CAR promoter gene that we have identified recently.
Finally, using chromatin immunoprecipitation, we demonstrated that the GR agonist dexamethasone induces histone
H4 acetylation at the proximal CAR promoter region, whereas LPS and IL-1β inhibit this acetylation. These data suggest that GR/NF-kB interaction converges at the level of
CAR transcription and involves chromatin remodeling.
The results of this study may provide a mechanistic explanation for the long standing observation that sepsis provoke drug metabolism inhibition. Moreover, as CAR controls the expression of several genes involved in bilirubin
elimination (the organic anion transporter SLC21A6,
which facilitates the enter of free-bilirubin in the hepatocyte, the phase II UDP-glucuronosyltransferase 1A1
(UGT1A1) protein , the principal means to glucuronidate
bilirubin, and the MRP2, which mediates the efflux of bilirubin diglucuronide across the apical membrane of the hepatocyte into the bile canaliculi), we hypothesized that NFkB-induced CAR deficit may contribute to inflammationinduced hyperbilirubinaemia , as observed recently in CAR
null mice.
TL11
TL10
PATHOPHYSIOLOGICAL
FACTORS AFFECTING CAR
GENE EXPRESSION
INDUCTION OF RAT CYP2B
GENES BY PHENOBARBITAL-LIKE INDUCERS : SOMETHING
OLD AND SOMETHING NEW
ALAN ANDERSON, MARIE-JOSÉE BEAUDET,
MAUD GRAVEL, AMAURY LACHAUD,
MARIANNE LAIZIER, and YANICK PAQUET
JEAN MARC PASCUSSI, PATRICK MAUREL, and
MARIE JOSÉ VILAREM MJ
INSERM 128, 1919 route de Mende, 34293 Montpellier,
France.
Centre de recherche, L’Hôtel-Dieu de Québec, 11 côte du
Palais, Québec G1R 2J6 and Département de biologie,
Université Laval, Québec G1K 7P4 Canada
[email protected]
The drug-metabolizing cytochrome P450 (CYP) enzymes are down-regulated during inflammation. This suppression can result in increased clinical toxicity of drugs.
Conversely, some drugs must be converted to their pharmacologically active or toxicologically active metabolites
by these enzymes, and suppression of their metabolism can
lead to a reduced therapeutic or toxic effect. Our resarch
has focused on the negative regulation of the orphan nuc-
Hepatic CYPs play a critical role in the metabolism of
hydrophobic xenobiotics1,2. The genes encoding these enzymes are either expressed constitutively or are induced by
various chemicals3. Much progress has been made concerning the molecular mechanism whereby phenobarbital
(PB) leads to the induction of the homologous rat CYP2B2
S52
and CYP2B1 and mouse Cyp2b10 genes4-9 and of the chicken CYP2H1 gene10. Although the chicken CYP2H1 gene is
PB-inducible in a chicken hepatoma cell line11, in the rodent system the only cultured cells in which CYP2B genes
respond normally to PB treatment are primary hepatocytes12,13. The PB response unit (PBRU), a 163-bp Sau3AI
fragment at coordinates -2317/-2155 in the CYP2B2
5’-flank, confers PB inducibility on heterologous promoters in primary rat hepatocytes and has the properties of
a transcriptional enhancer4,14,15. The homologous region of
the 5’-flank of the PB-inducible mouse Cyp2b10 gene contains a 162-bp segment with similar properties5; it is 92%
identical to the rat CYP2B2 PBRU15.
The rat CYP2B2 PBRU contains, among other putative
transcription factor recognition sites, three direct repeats
separated by 4 bp (DR-4), as well as an everted repeat separated by 7 bp (ER-7), of the nuclear receptor consensus
hexamer half-site motif AGGTCA. Two of the DR-4 sites,
NR1 and NR2, flank a nuclear factor 1 (NF1) site16 and
were recognized as putative nuclear receptor binding sites
by Negishi and coworkers in the homologous mouse
Cyp2b10 fragment16. The third DR-4 site, NR317, is upstream of NR1 and NR2. Negishi and coworkers also defined
a 51-bp PB responsive enhancer module (PBREM) within
the mouse Cyp2b10 PBRU sequence. The PBREM is limited to the NR1-NF1-NR2 elements and confers full PB responsiveness in primary mouse hepatocytes when placed directly adjacent to the heterologous tk promoter16. However,
replacing the PBRU with the PBREM in the CYP2B2 5’
flank, in the natural sequence context, reduces PB responsiveness in primary rat hepatocytes by at least four-fold6.
This and other evidence6 indicates that sequences outside
the PBREM are required for maximal PB responsiveness.
Possible candidates for such sequences are the upstream
NR3 site17 and the overlapping ER-7 site. Indeed mutational inactivation of the ER-7A half site, which is the same
sequence as the NR3B half site, reduces but does not abolish PB responsiveness; mutational inactivation of any
NR1 or NR2 half site has a similar effect6.
To determine which of NR3 or ER-7 are involved in
conferring PB responsiveness, their unique half sites were
mutated within the PBRU. The mutated plasmid reporter
constructs were then tested in the natural sequence context
for their ability to confer PB responsiveness in cultured
primary rat hepatocytes. The results indicate that NR3 is
the third element within the PBRU that is required to confer maximal PB responsiveness but the receptor that activates it remains to be discovered.
Negishi and coworkers18,19 have shown that there is
a PB-dependent nuclear accumulation of the constitutive
androstane receptor (CAR) in mouse liver. CAR, in the
form of a heterodimer with the retinoid X receptor (RXR)
binds to the retinoic acid β2 response element (βRARE)20,21
and to the NR1, NR2 and NR3 sites of the PBRU17,22.
CAR-RXR heterodimers activate transcription of reporter
genes driven by the PBREM and by oligomerized βRARE
and NR1 sequences in cultured cell lines9,18,19,22.
To assess the putative role of CAR-RXR binding to the
PBRU NR sites in conferring PB responsiveness, individual base pairs of the NR sites were changed and the effects on CAR-RXR binding were determined. The same
mutant sequences were incorporated into the NR sites within the PBRU and their effects on conferring PB responsiveness in luciferase reporter gene assays after transfection
into primary rat hepatocytes were evaluated. The results
suggest that something other than, or in addition to, CARRXR binding to the NR sites is required for maximal PB
responsiveness.
Mutational analysis of a newly-identified HNF4 element present in the PBRU was undertaken, using a reporter construct with the PBRU in the natural sequence context transfected into primary rat hepatocytes. The results
suggest that HNF4 acts as a negative regulator by competing for the binding to the NR1A half site thus preventing
normal transcriptional activation via the NR1 element. At
the NR2 site, mutation of the 4-bp spacer led to the loss of
a prominent doublet in EMSA assays. The proteins responsible for this doublet were identified by supershift analysis.
They were found to be Pbx1b-Prep1 and Pbx2-Prep1 heterodimers. The possible roles of HNF4 and Pbx-Prep1 as
negative regulators of PB responsiveness are presently under investigation.
The glucocorticoid receptor interacting protein 1 (GRIP1)
mediates PB-dependent nuclear translocation and activation of CAR in vivo but has been reported to have only modest effects in vitro9. Furthermore, normal PB responsiveness of CYP2B genes is not maintained in any known cell
line. In an effort to understand the molecular mechanisms
whereby PB induces CYP2B genes in vivo but not in cultured cells of hepatic origin, expression vectors for CAR,
RXR and GRIP1 were cotransfected into HepG2 cells
along with reporter constructs carrying the PBRU in different sequence contexts. CAR activated reporter gene transcription when the PBRU was juxtaposed to a basal promoter, but was essentially inactive when the PBRU was
tested in the natural sequence context. GRIP1 dramatically
increased CAR activation when tested with reporter constructs in which the PBRU was adjacent to the basal promoter and also, but to a much lesser degree, when the
PBRU was in its natural sequence context. It may be that
HepG2 and other cell lines lack as yet unidentified transcription factors that are necessary, in the natural sequence
context, for the PBRU to confer PB responsiveness on the
luciferase reporter.
Taken together, the results to be presented suggest that
something other than, or in addition to, CAR-RXR binding
to the NR sites is required for the rat CYP2B2 PBRU to
confer maximal PB responsiveness. Based on these results
as well as others to be discussed, the following model is
proposed to account for PB-dependent induction of the
CYP2B2 gene in primary rat hepatocytes. NR1 plus another
site, herein identified as NR3, are principal binding sites
for a PB-dependent transcription factor complex, presumably including CAR-RXR and as yet unknown factors.
S53
NR2 is an accessory site. At least two of these three sites
are required to confer a PB response. The maximal response is obtained when all three sites are functional and
HNF4 and Pbx-Prep1 are not bound to their cognate sites.
REFERENCES
1. Guengerich, F.P.: Mammalian Cytochromes P-450 Volume I. CRC Press, Boca Raton 1987.
2. Guengerich F.P., Shimada T.: Chem. Res. Toxicol. 4,
391 (1991).
3. Okey A.B.: Pharmacol. Ther. 45, 241 (1990).
4. Trottier E., Belzil A., Stoltz C., Anderson A.: Gene 158,
263 (1995).
5. Honkakoski P., Negishi M.: J. Biol. Chem. 272, 14943
(1997).
6. Paquet Y., Trottier E., Beaudet M.J., Anderson A.: J.
Biol. Chem. 275, 38427 (2000).
7. Zelko I., Negishi M.: Biochem. Biophys. Res. Commun. 277, 1 (2000).
8. Liu S., Rivera-Rivera I., Bredemeyer A.J., Kemper B.:
Biochem. Pharmacol. 62, 21 (2001).
9. Min G., Kemper J.K., Kemper B.: J Biol. Chem. 277,
26356 (2002).
10.Handschin C., Podvinec M., Looser R., Amherd R.,
Meyer U.A.: Mol. Pharmacol. 60, 681 (2001).
11. Handschin C., Meyer U.A.: J. Biol. Chem. 275, 13362
(2000).
12.Waxman D.J., Morrissey J.J., Naik S., Jauregui H.O.:
Biochem. J. 271, 113 (1990).
13.Sinclair P.R., Bement W.J., Haugen S.A., Sinclair J.F.,
Guzelian P.S.: Cancer Res. 50, 5219 (1990).
14.Park Y.K., Li H., Kemper B.: J. Biol. Chem. 271, 23725
(1996).
15.Stoltz C., Vachon M.-H., Trottier E., Dubois S., Paquet
Y., Anderson A.: J. Biol. Chem. 273, 8528 (1998).
16.Honkakoski P., Moore R., Washburn K.A., Negishi M.:
Mol. Pharmacol. 53, 597 (1998).
17.Kim J., Min G., Kemper B.: J. Biol. Chem. 276, 7559
(2001).
18.Honkakoski P., Zelko I., Sueyoshi T., Negishi M.: Mol.
Cell. Biol. 18, 5652 (1998).
19.Kawamoto T., Sueyoshi T., Zelko I., Moore R., Washburn K., Negishi M.: Mol. Cell Biol. 19, 6318 (1999).
20.Baes M., Gulick T., Choi H.S., Martinoli M.G., Simha
D., Moore D.D.: Mol. Cell Biol. 14, 1544 (1994).
21.Forman B.M., Tzameli I., Choi H.S., Chen L., Simha
D., Seol W., Evans R.M., Moore D.D.: Nature 395, 612
(1998).
22.Tzameli I., Pissios P., Schuetz E.G., Moore D.D.: Mol.
Cell Biol. 20, 2951 (2000).
S54
3
Invited Lectures – WL
LECTURES – WEDNESDAY – WL
trons from AdR, whereas CYP11A1-dependent substrate
conversion has been abolished. NMR studies of oxidised
and reduced Adx together with biochemical data lead to the
proposal of a new interaction mechanism among the three
proteins6. The NMR technique was also used to characterise in detail the function of a conserved histidine for redox-dependent dynamics and the interaction region between Adx and cytochrome c.
WL01 MITOCHONDRIAL
CYTOCHROME P450 SYSTEMS:
NEW INSIGHTS INTO
STRUCTURE AND FUNCTION
RITA BERNHARDT
REFERENCES
Institute of Biochemistry, Saarland University, P.O. Box 15
11 50, D-66041 Saarbrücken, Germany
[email protected]
1. Böttner, B., Schrauber, H., Bernhardt, R.: J. Biol.
Chem. 271, 8028 (1996).
2. Böttner, B., Bernhardt, R.:Endocrine Research 22, 455
(1996).
3. Bechtel S., Belkina, N., Bernhardt, R.: Eur. J. Biochem.
269, 1118 (2002).
4. Grinberg, A. V., Hannemann, F., Schiffler, B., Müller,
J.J., Heinemann, U., Bernhardt, R.: Proteins 40, 590
(2000).
5. Hannemann, F., Rottmann, M., Schiffler, B., Bernhardt,
R.: J. Biol. Chem. 276, 1369 (2001).
6. Beilke, D., Weiss, R., Löhr, F., Pristovsek, P., Hannemann, F., Bernhardt, R., Rüterjans, H.: Biochemistry
41, 7969 (2002).
In the human adrenal cortex glucocorticoids, mineralocorticoids, and androgens are being synthesized. Three mitochondrial cytochromes P450 are involved into this reaction cascade. The initial step is the side-chain cleavage of
cholesterol to form pregnenolone, catalyzed by CYP11A1.
The 11β-hydroxylase (P45011B1) is responsible for the
conversion of 11-deoxycortisol to cortisol. The aldosterone
synthase (CYP11B2) catalyses the 11β-hydroxylation,
18-hydroxylation and 18-oxidation of deoxycorticosterone
to form aldosterone.
Using site-directed mutagenesis in the C-terminal part
of human CYP11B1 and CYP11B2 (putative I-helix region)
we succeeded to change the regio- and stereoselectivity of
steroid hydroxylation by very few point mutations 1,2. Moreover, we were able to define the functional significance of
amino acid 112 in the putative substrate access channel of
human CYP11B2. Thus, we present the first example of an
attribution of substrate recognition and conversion to the
N-terminal part of human CYP11B23.
Mitochondrial cytochromes P450 get the electrons for
oxygen activation and following substrate conversion from
an iron-sulfur protein (adrenodoxin, Adx) of the [2Fe-2S]
type and a FAD-containing reductase (AdR). To study the
role of distinct amino acids of adrenodoxin in protein protein interaction and in electron transfer, mutants of adrenodoxin have been prepared by site-directed mutagenesis,
expressed in E. coli, and their structural and functional
properties have been characterised in detail over the past
10 years4. Different, but partially overlapping binding sites
for the partner proteins have been found and mutants with
improved electron transfer ability were obtained. A set of
mutants, where residues Glu39, Glu41 and Thr49 were replaced led to the suggestion that Adx possesses a second,
functionally relevant, binding site for the redox partners
being apart from the negatively charged region between
amino acids Glu65 and Asp79. This region is close to the
one, which in the homologous ferredoxin of P. putida, putidaredoxin, is responsible for the interaction with
CYP101. Moreover, calculation of the preferred electron
transfer pathways using the program HARLEM revealed
that this is the region with the highest conductivity for the
electrons5. In addition, a mutant lacking the entire interaction domain was shown of still being able to accept elec-
WL02 RESONANCE RAMAN AND
FEMTOSECOND COHERENCE
SPECTROSCOPY OF P450:
INTERACTIONS WITH
REDUCTASE, OXYGEN,
AND NITRIC OXIDE
P. M. CHAMPION
Physics Department, Northeastern University, Boston MA
02115 USA
Resonance Raman and femtosecond coherence spectroscopies are complementary spectroscopic methods that
operate in the frequency and time domains, respectively.
These methods can be used to monitor structure and dynamics of the heme and its ligands in order to reveal conformational substates and to prepare and monitor coherent states of a sample under reaction conditions1,2. The experiments discribed here focus on the nuclear motions associated with the heme proteins such as cytochrome P450, myoglobin, hemoglobin, and cytochrome c. Subsequent to photolysis of a sixth axial heme ligand, the (initially planar)
heme is left far from its final product state equilibrium
geometry. This leads to coherent oscillations of those modes composing the reaction coordinate for ligand binding
and dissociation. Analysis of the phase and amplitude of
these oscillations can help to determine the details of the
S55
reaction process. These studies, along with femtosecond
continuum measurements of the spectral dynamics and ligand binding kinetics as a function of temperature suggest
a general model for diatomic ligand binding in heme proteins.
REFERENCES
1. Sjodin T., Christian J., Macdonald I., Davydov R.,
Unno M., Sligar S., Hoffman B., Champion P.M.: Biochemistry 40, 6852 (2001).
2. Rosca F., Kumar A., Ionascu D., Ye X.,. Demidov A,. Sjodin T, Wharton D., Barrick D., Sligar S., Yonetani T,.
Champion P.M: J. Phys. Chem. A 106, 3540 (2002).
WL03 INTERACTIONS AMONG
REDUCTASE AND MULTIPLE
P450 ENZYMES IN MIXED
RECONSTITUTED SYSTEMS
WAYNE L. BACKES, RUSTY W. KELLEY
and DONGMEI CHENG
Louisiana State University Health Sciences Center, Department of Pharmacology, The Stanley S. Scott Cancer
Center, 533 Bolivar St., New Orleans, LA 70112 USA
[email protected]
Oxygenation reactions involving the cytochrome P450
system require interactions with other proteins to permit
the transfer of necessary electrons to the heme protein. The
flavoprotein NADPH-cytochrome P450 reductase (reductase) is essential for P450-dependent monooxygenase reactions, and has been shown to form a 1:1 molar complex
with P450 proteins (Miwa and Lu, 1984;Miwa and Lu,
1984;Miwa et al., 1979). Each of these proteins is associated with the microsomal membrane. Interestingly, there is
a large excess of P450 over the reductase, calling to question how the limiting concentrations of reductase can
supply the necessary reducing equivalents to the excess of
P450 molecules. Additionally, P450s exist in multiple
forms, with each form having its own substrate selectivity
and reductase binding characteristics. These characteristics
raise the question: „How are the proteins of the P450 system organized within the microsomal membrane?“ In an
effort to address this question, we have characterized the
interactions of microsomal proteins in reconstituted systems containing reductase and multiple P450 enzymes.
We have previously examined the potential for one
P450 to affect the catalytic behavior of another P450 isozyme. Interactions between P450s were demonstrated by
comparing the catalytic behavior of CYP2B4 and CYP1A2
in simple and mixed reconstituted systems. Two substrates
that are preferentially metabolized by CYP2B4 (benzphe-
S56
tamine and 7-pentoxyresorufin (7PR)) were used to demonstrate the interaction. Whereas benzphetamine demethylation showed a small increase in catalytic activity when
both CYP1A2 and CYP2B4 were present in the reconstituted system, CYP2B4-dependent 7-pentoxyresorufin-O-dealkylation (PROD) was found to be dramatically inhibited
by the inclusion of CYP1A2 (Cawley et al., 1995). Interestingly, this inhibition of PROD was much larger when reductase levels were subsaturating. The results are consistent with the formation of a high affinity complex between
CYP1A2 and reductase that prevents reductase binding and
electron transfer to CYP2B4, particularly when reductase
levels are subsaturating. Kinetic analysis of PROD activity
at a series of reductase concentrations showed the system
to be consistent with the formation of heteromeric
CYP2B4-CYP1A2 complexes. In the presence of 7PR, the
CYP1A2 moiety of the complex has an increased affinity
for reductase, effectively drawing the reductase away from
CYP2B4 and inhibiting this CYP2B4-dependent activity
(Backes et al., 1998). Similar interactions have also been
observed in rabbit liver microsomes. Rabbits were treated
with either phenobarbital (PB), β-naphthoflavone (βNF), or
both inducers to enrich the microsomes with CYP2B4,
CYP1A2, and both enzymes, respectively. The relative rates of benzphetamine demethylation and PROD were similar to those found in the corresponding reconstituted systems (i.e. benzphetamine metabolism was stimulated in
rabbits treated with both inducers, whereas PROD was inhibited). These results suggest that the interactions observed in the reconstituted systems are not an artifact of the
reconstitution process, but are also observed under the
more natural conditions of the microsomal membrane
(Cawley et al, 2001).
Several approaches have been used in an effort to characterize the interactions among these proteins. The effects
of lipid concentration on reductase binding were examined
in simple reconstituted systems containing only a single
P450, and in mixed reconstituted systems containing both
CYP1A2 and CYP2B4. Although the apparent affinity for
reductase-CYP2B4 was not significantly affected, there
was a 90% decrease in CYP2B4-dependent activities at saturating reductase when the lipid:P450 ratio was increased
10 fold. Interestingly, CYP1A2 under the same conditions
showed only a modest decrease (~30%) in activity at high
lipid concentrations. Again, the apparent affinity of reductase for the P450 was not substantially affected by lipid
concentration.
We also examined the potential for the complex between CYP1A2 and CYP2B4 to be mediated by ionic interactions. The effect of ionic strength on PROD was examined using reconstituted systems containing reductase/CYP2B4, reductase/CYP1A2 and the mixed reconstituted system (CYP1A2/CYP2B4/reductase). PROD in each
reconstituted system was affected by ionic strength, reaching a maximum at 50 mM sodium phosphate (pH 7.4).
The activities continued to decrease at buffer concentrations
of 75 mM, 200 mM and 500 mM. At low ionic strength,
PROD activities in the mixed reconstituted systems were
lower than observed for CYP2B4/reductase binary system,
consistent with the interaction between CYP1A2 and
CYP2B4. Interestingly, as the phosphate concentration was
increased above 75 mM, inhibition of PROD in the mixed
reconstituted system was no longer observed. In fact, at
500 mM phosphate, a synergistic response was obtained.
These results are consistent with complex formation between CYP2B4 and CYP1A2 being mediated by ionic interactions.
The stability of the binary and mixed reconstituted systems was also examined. In these studies, reconstituted
systems were prepared and examined after preincubation
for up to 4 days. CYP1A2-dependent PROD was not diminished by preincubation, even exhibiting a small increase
after preincubation for 4 days. In contrast, CYP2B4 activity showed a continual decline with each day of incubation. After preincubation of the reconstituted system for 4
days there was a 65% decrease in CYP2B4-dependent
PROD. Interestingly, in the mixed reconstituted system,
there was a continual increase in PROD activity with prolonged incubation times. When comparing CYP2B4/reductase and the CYP2B4/CYP1A2/reductase reconstituted
systems, the characteristic inhibition of PROD in the mixed reconstituted system was no longer observed after preincubation for 4 days. These results are consistent with
a slow reorganization of the proteins in the reconstituted
system with time. (Supported in part by NIEHS 04344, and
the Stanley S. Scott Cancer Center).
REFERENCES
1. Backes WL., Batie C. J., Cawley G. F.: Biochem 37,
12852 (1998).
2. Cawley GF., Batie C. J., Backes W. L.: Biochem 34,
1244 (1995).
3. Cawley GF., Zhang S., Kelley R. W., Backes W. L.:
Drug Metab Dispos 29, 1529 (2001).
4. Miwa GT., Lu A. Y. H.: Arch Biochem Biophys 234,
161 (1984).
5. Miwa GT., West S. B., Huang M. T. Lu A. Y. H.: J Biol
Chem 254, 5695 (1979).
S57
WL04 EXPERIMENTAL IN VITRO
MODELS OF DRUG
METABOLISM
ment with commercially available preparations consisting
of a defined human enzyme or enzymes is inevitable. Although an ideal animal model apparently does not exist, the
known similarities between P450s of animal and human
origin may however be used in rationalizing the possible
use of cells (hepatocytes) for construction of bioartificial
liver-supporting devices.
PAVEL ANZENBACHERa, EVA ANZENBACHEROVÁb,
LUCIE KOUSALOVÁa, and JI¤INA MARTÍNKOVÁc
Acknowledgment.
The authors thank the COST B 15.50 project for
support.
a
Institute of Pharmacology and bInstitute of Medical Chemistry and Biochemistry, Faculty of Medicine, Palacky
University, Hnûvotínská 3, CZ-775 15 Olomouc, Czech Republic, [email protected], bInstitute of Pharmacology,
Faculty of Medicine at Hradec Králové, Charles University, ·imkova 870, CZ-500 01 Hradec Králové, Czech
Republic
REFERENCES
1. Zuber R., Anzenbacherová E., Anzenbacher P.: J. Cell.
Mol. Med. 6, 189 (2002).
2. Soucek P., Zuber R., Anzenbacherová E., Anzenbacher
P., Guengerich F.P.: BMC Pharmacology 1, 11 (2001).
3. Olsen A., Hansen K.T., Friis C.: Chem.-Biol. Interact.
107, 93 (1997).
4. Anzenbacher P., Souãek P., Anzenbacherová E., Gut I.,
Hrub˘ K., Svoboda Z., Kvûtina J.: Drug Metab. Disposition 26, 56 (1998).
5. Roussel F., Duignan D.B., Lawton M.P., Obach R.S.,
Strick C.S., Tweedle D.J.: Arch. Biochem. Biophys.
357, 27 (1998).
6. Nedelcheva V., Gut I.: Xenobiotica 24, 1151 (1994).
7. Sharer J.E., Shipley L.A., Vandenbranden M.R., Binkley S.N., Wrighton S.A.: Drug Metab. Disposition 23,
1231 (1995).
The search for an ideal experimental model for in vitro
studies on drug metabolism has continued since pharmacology was established as a respected discipline of medicine.
Most recently, an increasing knowledge on properties of
individual drug metabolizing enzymes in various species of
experimental animals contributed significantly to understanding of the interspecies differences and to better choice
of a species suitable for a given type of experiment. For
example, the rat and the systems derived from rat are used
now mostly as a first attempt to get the basic information
on possible metabolic routes. Here, the experiments at the
level of microsomal fraction are performed first. When an
involvement of cytochromes P450 in metabolism of a drug
is found, then the more reliable models should be used taking advantage from known properties of substrate specifity of cytochrome P450 forms in respective animal species1. Here, the more detailed study requires a model which
can give answers on the role of a particular form of cytochrome P450 enzyme. The CYP3A29 form of pig or minipig is most probably the closest counterpart of the human
CYP3A4 enzyme2. The studies with minipig or pig hepatocytes3, microsomes4 as well as with the reconstituted CYP
systems2 indicate their suitability in following the processes mediated by this CYP3A subfamily. For metabolic
transformations dependent on CYP2D, the dog seems to
give the results5 close to those obtained with human
CYP2D6. The CYP2C family of rat does not seem to be
a good model of the human counterpart as it has different
substrate specificities6. Hence, the results obtained with
mouse models where CYP2C enzymes were found to participate should be taken with care. For CYP2C, the most
suitable model seems to be the Rhesus monkey7. The
CYP1A2 and CYP2E1 enzymes of man are very close in
their properties to those of other species thus allowing their
use in studies on drug metabolism.
Hence, it can be concluded that there is not an ideal
single experimental animal model system available even
when properties of many human CYP enzymes are close to
those of the respective models. For studies on biotransformation of a compound, the results obtained with animal based systems are of a preliminary character. An experi-
WL05 SHOULD WE PERSONALIZE
DRUG THERAPY ACCORDING
TO CYTOCHROME P450 GENOTYPE AND/OR PHENOTYPE?
UWE FUHR
Institute for Pharmacology, Clinical Pharmacology,
University of Köln, Gleueler Strasse 24, 50931 Köln,
Germany, [email protected]
Large prospective randomized double-blind clinical
trails are the gold standard to assess safety and efficacy of
drug therapy. However, these studies are measuring average drug effects, and even if a clear benefit is observed
based on clinical endpoints, there are individuals with
a lack of therapeutic effect, and there are patients with an
excessive drug response. More than 5 % of in-patients suffer from serious adverse drug reactions1, and it has been
estimated that at least half of these reactions could have
been avoided by appropriate selection of drug and/or drug
dose. Thus, a higher degree of personalization in drug therapy is expected to considerably improve the risk/benefit
ratio.
S58
Main reasons for the interindividual heterogeneity in
drug response include heterogeneity in the disease(s) treated, in drug targets, in comedication, and/or in drug concentrations. Heterogeneity attributable to drug concentrations may be handled by a personalized dose adjustment.
This may be done based on clinical response, on drug concentration (=therapeutic drug monitoring), or an factors
known to predict clinical response and/or drug concentration.
Cytochrome P450 enzymes (CYPs) mediate rate-limiting steps of inactivation (or activation) of many important
drugs and show pronounced interindividual differences in
activity. It is reasonable to assume that individual activity
of the respective CYP may have a major impact on the dose
vs. response relationship of a drug. If this CYP is genetically polymorphic, the individual genotype may be the major factor determining enzyme activity. However, it is obvious that CYP polymorphisms and/or CYP activity is several levels of hierarchy away from drug response, and
only if its impact is major on all subsequent levels, individual CYP activity is expected to be relevant for therapeutic outcome.
Several decades ago, it has been recognized that individual CYP activity (at this time not identified as such) may
be related to differences in drug effects. During the last decade, extensive research has been carried out in order (i) to
identify variant CYP genes related to aberrant activity, (ii)
to identify enzymes involved in the metabolism of individual drugs, (iii) to assess frequencies of CYP alleles in different populations, (iv) to quantify the impact of CYP variants on pharmacokinetics of individual drugs, and (v) to
develop methods for CYP phenotyping. As a result, e.g. today genotyping identifies >99 % of CYP2D6 poor metabolizers2, individual elimination rates of tolbutamide may be
derived from the contribution of individual CYP2C9 alleles3, or 2/3 of the variation in docetaxel pharmacokinetics
may be explained by individual CYP3A4 activity4. Furthermore, pharmaceutical companies incorporated CYP genotyping and CYP phenotyping in the drug development
process.
Fewer studies have been conducted to relate CYP activities to drug effects in patients. Most of such studies are
retrospective and have been done in small populations
only. The few examples for prospective investigations include the assessment of the relationship between CYP2D6
genotype to efficacy and tolerability of haloperidol5, or to
the antiemetic effects of 5-hydroxytryptamine type 3 receptor antagonists6. A quantitative proposal for dose adjustment as it has been made for antidepressants based on
CYP2D6 and CYP2C19 genotype is a rare exception7. No
appropriate clinical studies clearly assessing the therapeutic benefit of dose personalization based on CYP activity
are available to my knowledge. As a consequence, information on individual CYP activity currently is not being
applied to personalize drug therapy. Because of the lack of
data, despite its potential such procedures to date cannot be
recommended outside clinical studies.
CYP genotyping has become increasingly reliable, simple and cheap, e.g. by light cycler and gene chip technologies. Likewise, CYP phenotyping made major progress,
mainly by increased analytical sensitivity brought about by
LC-MS/MS with the possibility to administer very low doses of marker drugs. The tools for assessment of individual
activity of human CYPs with outstanding relevance for
drug metabolism, i.e. CYP3A4, CYP2D6, CYP2C9,
CYP1A2 and CYP2C19, are available. What is needed are
large prospective randomized studies comparing traditional
dose adjustment based on clinical effects only with additional dose personalization based on CYP activity. In these
trials, both average efficacy / tolerability and the degree of
interindividual variation in these aspects should be compared. From such studies, algorithms and software tools (expert systems) need to be derived to take CYP activity into
account for dose calculation. Finally, the cost-effectiveness
of the approach must be addressed. Only then, it will be
possible to judge whether CYP genotyping and/or phenotyping is worthwhile to be considered in clinical medicine.
REFERENCES
1. Lazarou J., Pomeranz B.H., Corey P.N.: JAMA 279,
1200 (1998)
2. Griese E.U., Zanger U.M., Brudermanns U., Gaedigk
A., Mikus G., Mörike K., Stuven T., Eichelbaum M.:
Pharmacogenetics 8, 15 (1998)
3. Kirchheiner J., Bauer S., Meineke I., Rohde W., Prang
V., Meisel C., Roots I., Brockmöller J.: Pharmacogenetics 12, 101 (2002)
4. Hirth J., Watkins P.B., Strawderman M., Schott A.,
Bruno R., Baker L.H.: Clin Cancer Res 6, 1255 (2000)
5. Brockmöller J., Kirchheiner J., Schmider J., Walter S.,
Sachse C., Müller-Oerlinghausen B., Roots I.: Clin
Pharmacol Ther 72, 438 (2002)
6. Kaiser R., Sezer O., Papies A., Bauer S., Schelenz C.,
Tremblay P.B., Possinger K., Roots I., Brockmöller J.:
J Clin Oncol 20, 2805 (2002)
7. Kirchheiner J., Brøsen K., Dahl M.L., Gram L.F., Kasper S., Roots I., Sjøqvist F., Spina E., Brockmöller J.:
Acta Psychiatr Scand 104, 173 (2001)
S59
WL06 IN VITRO TO IN VIVO
CORRELATIONS - A CRITICAL
ASSESSMENT OF VARIOUS
APPROACHES TO MEASURE
DRUG METABOLISM AND
METABOLIC INTERACTIONS
bitors, quantitation of CYPs by Western blotting). Other
experimental in vitro systems include liver slices, cultured
hepatocytes, recombinant expressed enzymes, and permanent cell lines. Currently in silico models are being increasingly used for predicting metabolism and interactions.
During the development of an NCE, at least the following investigations are usually required, the earlier the
better:
- Measurement of the disappearance of a chemical in human liver preparations; „metabolic stability“
- Identification of principal metabolites; „metabolite profile and proposed metabolic tree of a chemical“
- Development of an analytical „routine“ method for metabolites
- Identification of enzymes catalysing metabolic routes
with the aid of diagnostic inhibitors and antibodies, recombinant enzymes and correlation studies
- Enzyme kinetic characterisation of principal metabolic
reactions; for scaling up and predicting in vivo kinetics
of an NCE
- Affinity of a chemical (and/or its principal metabolites)
for CYP (or other) enzymes; for predicting potential metabolic interactions
OLAVI PELKONEN, JOUKO UUSITALO, and MIIA
TURPEINEN
University of Oulu, Department of Pharmacology and Toxicology, POB 5000, FIN-90014 Oulu, Finland,
[email protected]
Elucidation of metabolic stability, metabolic routes, metabolising enzymes and their kinetics, and metabolic interactions is obviously important for any chemicals to which humans are exposed. It is even more important for pharmaceuticals, because this information is needed for selecting leads
and candidate drugs during drug discovery and development. In short, metabolism determines to a great extent the
pharmacokinetic properties of most drugs and is often behind interactions, metabolic idiosyncrasies and so on2, 3.
Human-liver derived in vitro systems are thought to be
useful in early studies of metabolic stability, in identifying
enzymes capable of metabolising, and interacting with,
drugs in use or under development (NCE, new chemical
entity) and several of these systems are currently under validation as to their predictive power to the in vivo situation4. These approaches are expected to provide crucial information for more in-depth studies during the preclinical
and clinical phases of drug development. Some examples
of the approach have been published1, 5.
The most important goals for using various human liver-derived in vitro systems include the following:
1) to elucidate and determine principal metabolic routes of
an NCE and to tentatively identify principal metabolites;
2) to identify, with human liver homogenate or microsomal preparations, the CYP enzymes catalysing the principal oxidation routes (primary metabolites) and to gain
some quantitative data on their significance for the overall metabolic fate of an NCE;
3) to provide useful background information for characterising potential interactions and physiological, genetic
and pathological factors affecting the kinetics and variability of an NCE in in vivo situation.
Some typical experiments and their outcomes
Effects of various diagnostic CYP-selective inhibitors
on NCE metabolism: an estimation of the role of each CYP
enzyme to contribute to the metabolism of a chemical. On
the basis of this information, it is possible to perform predictive calculations to the in vivo behaviour of a chemical.
Ability of recombinant enzymes to catalyse metabolite
formation of an NCE: gives a direct indication of the potential of each recombinant CYP to catalyse the reaction.
However, there is still somewhat uncertain of whether the
results from recombinant enzymes could be extrapolated to
the whole microsomes and to the in vivo situation.
Correlation of CYP-selective model activities with metabolite formation of an NCE: gives an indirect indication
of the participation of each CYP enzyme to catalyse the reaction. The results are, however, confirmatory and should
be evaluated in the context of other approaches.
After the above studies, it is possible to plan further
preclinical, molecular, toxicological and clinicopharmacological studies, with focussed consideration of those CYP
enzymes which are of importance for the metabolism and
kinetics of an NCE.
Validation of various approaches
In vitro approaches for predicting in vivo behaviour of
pharmaceuticals have been developing for quite some time,
but still there are relatively few attempts to validate these
approaches. The validation involve several steps: test development, pre-validation, validation, independent assessment
and regulatory acceptance if appropriate. With respect to the
methods described above, the selection of pre-validation and
validation substances, in addition to proper substrates and
inhibitors, is of utmost importance. Because ultimately cli-
Experimental methods available currently
Human liver samples obtained under proper ethical permission have been used as a golden standard. These preparations have usually been extensively characterised to be
used for the primary screening (sufficient model activities,
no known polymorphisms, expected effects of model inhi-
S60
nical significance is one of the more important goals, proper
consideration should be given to the selection of selectivity,
specificity and potency of various substances used, which
should preferably be clinically used drugs, either currently
or in the past, so that enough proper in vivo information is
available for the assessment of prediction models. The prediction model itself should be adequately characterised with
respect to conversion factors (e.g. what is the „standard“
amount of microsomal protein in the unit of liver weight)
and algorithms (e.g. what kind of values should be assumed
for e.g. volume of distribution or renal clearance, if not
known). These validation requirements mean that the process of validation is a rather cumbersome and time-consuming undertaking (and is regarded by some as a „dull“ exercise) and not very often performed in this field.
dies have shown that the chemosensitivity of tumors to
P450 prodrugs can be dramatically increased by transfer of
a rodent or human CYP gene, which confers on the target
tumor tissue the capacity for localized prodrug activation
and, in the case of cyclophosphamide, induces a mitochondrial/caspase 9-dependent pathway of apoptotic cell
death. This P450 gene-directed enzyme prodrug therapy
(P450 GDEPT) greatly enhances the therapeutic effect of
P450-activated anti-cancer prodrugs without increasing
host toxicity associated with systemic distribution of active
drug metabolites formed by the liver. The efficacy of P450
GDEPT can be augmented in several ways:
1) by further increasing the partition ratio for tumor:liver prodrug activation in favor of increased intratumoral
metabolism, e.g., by co-expression of the flavoenzyme
NADPH-P450 reductase, by localized prodrug delivery or
by the selective pharmacologic inhibition of liver prodrug
activation;
2) by anti-angiogenic scheduling of prodrug administration, which substantially magnifies the anti-tumor effect
and leads to major regression of established tumors; and
3) by co-expression of an anti-apoptotic factor, which
inhibits tumor cell apoptosis in a manner that prolongs the
generation of soluble, bystander cytotoxic metabolites but
does not ultimately block tumor cell death. P450 GDEPT
prodrug substrates are diverse in their structure, mechanism of action and choice of optimal prodrug-activating
P450 gene; they include both established and investigational anti-cancer prodrugs, as well as bioreductive drugs that
can be activated by P450/P450 reductase in a hypoxic tumor environment. Several strategies have now been employed to achieve the tumor-selective gene delivery that is
required for the success of P450 GDEPT; these include the
use of tumor-targeted cellular vectors and oncolytic viruses
that replicate in a tumor-selective manner. Overall, P450based GDEPT presents several important, practical advantages over other GDEPT strategies that may facilitate the
incorporation of P450 GDEPT into existing cancer treatment regimens. Recent reports of clinical efficacy in P450based phase I/II gene therapy trials for breast and pancreatic cancer patients support this conclusion.
Supported in part by NIH grant CA49248 (to D. J. W.)
REFERENCES
1. Andersson TB, Sjöberg H, Hoffmann K-J, Boobis AR,
Watts P , Edwards RJ, Lake BG, Price RJ, Renwick AB,
Gómez-Lechón MJ, Castell JV, Ingelman-Sundberg M,
Hidestrand M, Goldfarb PS, Lewis DFV, Corcos L, Guillouzo A, Taavitsainen P, Pelkonen O.: Drug Metab
Disp 2001; 29, 712.
2. Boobis AR, Kremers P, Pelkonen O, Pithan K (editors):
EUR 18569 - European Symposium on the Prediction of
Drug Metabolism in Man: Progress and Problems. Luxembourg: Office for Official Publications of the European Communities. 332 pp. 1999.
3. Pelkonen O, Baumann A, Reichel A. Pharmacokinetic
Challenges in Drug Discovery. Ernst Schering Research
Foundation Workshop 37. Springer, Berlin, 2002.
4. Pelkonen O, Mäenpää J, Taavitsainen P, Rautio A, Raunio H.: Xenobiotica 28, 1203, 1998.
5. Pelkonen O, Myllynen P, Taavitsainen P, Boobis AR,
Watts P, Lake BG, Price RJ, Renwick AB, Gómez-Lechón MJ, Castell JV, Ingelman-Sundberg M, Hidestrand
M, Guillouzo A, Corcos L, Goldfarb PS, Lewis DFV:
Xenobiotica 2001, 31, 321.
WL07 P450, ANTI-CANCER DRUGS
AND GENE THERAPIES
REFERENCES
1. Chen L., Waxman D. J.: Curr Pharm Des 8, 1405
(2002).
2. Jounaidi Y., Waxman D. J.: Cancer Res 61, 4437
(2001).
3. Schwartz P. S., Chen C. S., Waxman D.J.: Cancer Res
62, 6928 (2002).
DAVID J. WAXMAN, YOUSSEF JOUNAIDI,
CHONG-SHENG CHEN, HONG LU, TING SU, and
PAMELA S. SCHWARTZ
Dept. of Biology, Boston University, Boston MA 02215
Several commonly used cancer chemotherapeutic prodrugs, including cyclophosphamide and ifosfamide, are activated via a liver cytochrome P450 (CYP)-catalyzed prodrug activation reaction. Rodent and human hepatic P450s
display clear similarities in their catalytic specificities towards these anti-cancer prodrug substrates. Preclinical stu-
S61
14α-DM and to assign the exact function to the CYP51
structural regions. Our preliminary studies have already established the essential role of conserved residues in of the
three N-terminal SRSs of P450s (B’helix/BC loop, helix
F and helix G) and led to the experimentally supported hypothesis that significant conformational rearrangements in
MTCYP51 occur upon substrate binding (presumably closing BC loop, which brings it to the position similar to that
found in substrate bound P450 structures for example,
P450BM33 and movement of helices F and G towards the
heme, resembling that shown in P450cam after interaction
with Ru-linker substrates4).
As the studies progress new CYP51 sequences will appear from genomic sequencing which will prove useful in
understanding CYP51 structure/function. Newly reported
CYP51 sequences in the Trypanosoma family (Trypanosoma brucei and Trypanosoma cruzi) indicate that unlike
the other reported CYP51 sequences, certain residues in
the N-terminal SRSs are not conserved. This provides an
additional opportunity to understand the role of the highly
conserved residues in CYP51 function. This presentation
will summarize our studies aimed at identifying the structural requirements for sterol 14α-demethylation.
WL08 CYP51: STRUCTURE,
FUNCTION, EVOLUTION
GALINA LEPESHEVA, LARISSA PODUST, and
MICHAEL R. WATERMAN
Department of Biochemistry, Vanderbilt University School
of Medicine, Nashville, TN 37232 USA, [email protected]
CYP51 (sterol 14α-demethylase) is an essential enzyme
in sterol biosynthetic pathways and the only cytochrome
P450 gene family having catalytically identical homologs in
different phyla. It is required for conversion of lanosterol to
cholesterol (animals), 24-methylenedihydrolanosterol to ergosterol (fungi/yeast) and obtusifoliol to phytosterols
(plants). It is the most widely distributed member of the cytochrome P450 superfamily, also being found in lower eukaryotes and some bacteria and is perhaps the most ancient of
P450s. All forms catalyze 14α-demethylation (14α-DM) of
∆8-sterols through a catalytic cycle involving three successive monooxygenation steps. Also, CYP51s are known to
have high affinity for azole inhibitors and are primary targets
for development of drugs for treatment of different fungus infections. Thus CYP51 is a particularly important member of
the cytochrome P450 superfamily because of its potential for
providing detailed understanding of structural requirements
for substrate orientation and for its important role as a drug
target in human infections. We have determined the high resolution X-ray structure of the soluble form of CYP51 from
Mycobacterium tuberculosis (MT)1, 2. This structure has allowed us to assign the location of secondary structural elements
to other family members with high probability.
Across different phyla, CYP51 has relatively low sequence identity (22-32%). Yet all members metabolize only
an extremely limited set of substrates, catalyzing the first
step in sterol biosynthesis following cyclization. Alignment of the more than 50 known CYP51 sequences throughout biology reveals the presence of ~40 conserved amino
acids. Three are the heme-ligated Cys and the conserved
EXXR in the K-helix. We propose that included within
these are the main structural requirements for 14α-DM,
allowing the enzyme to catalyze the same reaction in organisms which are evolutionarily related over hundreds
of millions of years. Many of these residues are located
within the secondary structure elements designated as substrate recognition sequences (SRS) of P450s, supporting
the notion of their essential role in 14α-DM. By site-directed mutagenesis of both MT and human CYP51 followed
by detailed biochemical and biophysical analysis of the
mutants we are identifying the role of conserved residues
in substrate binding and enzymatic activity, protein-protein
interactions and electron transfer, and maintenance of
3-D structure and protein folding. Connection between
the effect of mutagenesis and the location of each residue
will make it possible to predict the role of each in sterol
REFERENCES
1. Podust L.M., Poulos T.L., Waterman M.R.: Proc. Natl.
Acad. Sci. USA, 98, 3068 (2001).
2. Podust L.M., Stojan J., Poulos T.L., Waterman M.R.:
J. Bioinorg. Chem. 87, 227 (2001).
3. Haines D.C., Tomchick D.R., Machius M., Peterson
J.A.: Biochemistry 40, 13456 (2001).
4. Dunn A.R., Dmochowski I.J., Bilwes A.M., Gray H.B.,
Crane B.R.: Proc. Natl. Acad. Sci. USA 98, 12420
(2001).
WL09 LANOSTEROL
14α-DEMETHYLASE,
CHOLESTEROL AND
REPRODUCTION
DAMJANA ROZMAN, MARKO COTMAN,
KLEMENTINA FON TACER, and MARTINA FINK
Institute of Biochemistry, Medical Center for Molecular
Biology, Faculty of Medicine, University of Ljubljana, Slovenia
Lanosterol 14α-demethylase (CYP51) is a microsomal
enzyme that in the presence of NADPH cytochrome P450
reductase, NADPH and molecular oxygen removes the
14α-methyl group from lanosterol in a complex, threestep reaction. The final reaction product is 4,4,-dimethyl5α-cholesta,8,14,24-dien-3β-ol, known also as folicular
S62
Enzymatic activity and synthesis of MAS
Beside on the ER, a complete lanosterol 14α-demethylase system (CYP51 and its redox partner NADPH-P450
reductase) is located also on acrosomal membranes and remains there stabilized for several days during sperm maturation phases. CYP51 enzymatic activity (conversion of lanosterol to FF-MAS) is detected in the liver microsomes of
all tested mammals as well as in testis/isolated germ cells
of rat, mouse and ram. Isolated acrosomal membranes of
bull sperm as well as sperm homogenates of bull and ram
also seem to harbor CYP51 enzymatic activity. FF-MAS,
a direct product of the CYP51 enzymatic reaction, is not
detectable in livers of adult (65-70 day-old) or pre-pubertal (21-23-day old) male mice, but is easily detectable in
the tesits. Same applies to the testis meiosis activation sterol T-MAS. Besides in the testis, MAS are detectable also
in brain, suggesting a similar regulatory mechanisms of
cholesterol biosynthesis in both organs.
In conclusion, transcription of the mammalian CYP51
mRNA, CYP51 protein intracellular trafficking and likely
also post-translational modification, differ between liver
and testis (male germ cells). While CYP51 in the liver serves mainly to contribute to production of cholesterol, it is
believed that CYP51 features in male egrm cells contribute
majorly to production of MAS. MAS that were shown to
re-initiate meiosis of mouse oocytes in vitro have yet not
completely understood roles in mammalian reproduction in
vivo.
fluid meiosis activating sterol (FF-MAS). FF-MAS and TMAS (testis meiosis activating sterol), the product of the
subsequent reaction in the cholesterol biosynthesis pathway, acumulate in female and male gonads, respectively.
MAS stimulate the reinitiation of meiosis in mouse oocyte in vitro and are belived to have important roles in
fertilisation [Byskov, 1995 #71]. In contrast to gonadal
tissues, MAS are not easily detectable in the liver where
they represent intermediates in cholesterol biosinthesis1.
Biochemical mechanisms contributing to MAS accumulation in gonads are not yet understood. It is, however, believed that events at transcriptional and post-transcriptional levels contribute to dis-regulation of the cholesterol
biosynthesis pathway, leading consequently to accumulation of MAS.
Transcription
Different transcription factors and coactivator proteins
contribute to formation of transcriptional complexes at
CYP51 promoter in liver and testis. In the liver,
SREBP1a (immortal cell lines) or SREBP2 (tissue) are major transactivators that, however, require a cAMP-dependent transcription factor for optimal transactivation2. In the
testis (male germ cells) CREMτ is the major transactivator
but a germ cell-specific transcription factor SREBP-2gc
might also be involved in CYP51 activation. Not only
CYP51, also other cholesterogenic genes seem to be regulated in a different manner in liver compared to the testis.
We have proposed earlier that a discordant regulation of
the cholesterol biosynthesis pathway in the testis is the first
regulatory level leading to accumulation of intermediates
of cholesterol biosynthesis MAS1.
REFERENCES
1. Fon Tacer K., Haugen T. B, Baltsen M., Debeljak N.,
Rozman D.: J. Lipid. Res. 43, 89 (2002).
2. Halder S., Fink M., Waterman M., Rozman D.: Mol.
Endo. 16, 1853 (2002) .
Intracellular protein transport
Smooth endoplasmic reticulum of mouse liver contains
a 53 kDa CYP51. Significant amount of this protein is detected also in cis Golgi but not in trans Golgi, suggesting
retreival of CYP51 from Golgi back to the ER in the liver.
A 53 kDa CYP51 is the major form also in mouse male
germ cells where it resides in ER, Golgi and a male germ
cell-specific Golgi-derived organelle acrosome. CYP51 remains detectable during all phases of acrosome development (15 days in the mouse), including most mature spermatids that lack ER and Golgi, indicating that in male germ
cells CYP51 trafficks from ER through Golgi to its final
destination acrosome. Detection of a 50 kDa CYP51 in
acrosomal membranes of ram and bull ejaculated sperm
suggests a conserved mechanism of CYP51 traffic in mammalian germ cells. The appearance of high molecular mass
(HMM) CYP51-immunoreactive proteins is also a common feature. HMM CYP51s are detected in ram and bull
acrosomal membranes and in Golgi-enriched fractions of
mouse liver microsomes, suggesting that post-translational
modifications leading to HMM compelxes arise in the
Golgi. This is a first example of a cytochrome P450 protein
with different intracellular trafficking and subcellular localization, depending on a cell type.
WL10 P450 HUMANIZED MICE:
NOVEL TOOLS FOR THE STUDY
OF DRUG AND CARCINOGEN
METABOLISM, AND THE
SEARCH FOR ENDOGENOUS
HUMAN P450 SUBSTRATES AND
FUNCTIONS
FRANK J. GONZALEZa, AIMING YUa,
CONNIE CHEUNGa, CAMILLE P. GRANVILa,
ADRIAN KUPFERb, and JEFFREY R. IDLEb
a
Laboratory of Metabolism, National Cancer Institute, Bethesda, Maryland 20892, U.S.A., bUniversity of Bern, Bern,
Switzerland
There are marked species differences in the expression
and catalytic activities of P450s that metabolize xenobio-
S63
tics. This is especially notable between mice and rats, commonly used experimental models, and humans. P450 humanized mice were developed using and λ phage, BAC and
PAC genomic clones as transgenes. Humanized lines expressing CYP1A1/CYP1A2, CYP1B1, CYP2D6, CYP2E1,
CYP3A4 lines were characterized and found to accurately
express human P450 catalytic activities at levels comparable to or higher than the corresponding activities found in
human tissues. New highly sensitive LC-MS/MS-based assays were developed to perform pharmacokinetic studies
using a single mouse and non-radiolabeled drugs. These
novel mouse lines offer the opportunity to predict human
drug and carcinogen metabolism and disposition, and to search for endogenous substrates for human P450s.
The results so far accumulated indicate: (1) The functional zonation of the adrenal cortex is governed by the expression of each terminal steroidogenic enzyme such as
P450aldo in zG and P45011β in zFR2. (2) Both functional
and morphological zonation are recognized in the adrenal
gland of adult rats, but not in that at fetal stages 3. The zones were established in the third week after birth. (3) The
adrenal gland after the enucleation can regenerate the entire adrenal cortex with both functional and morphological
zonation3, suggesting that the zonation is an inherent nature
of the adrenocortical cells. (4) The fourth zone is present in
the adult adrenal cortex as a thin cell layer between zG and
zF4. The cells there do not express P450aldo, P45011β nor
P45017α,lyase, an androgen-synthesizing enzyme. In other
words, the zone lacks any differentiated function of adrenocortical zones. Accordingly it was named „the undifferentiated cell zone (zU)“. (5) zU was shown to be the sites
of cell proliferation under the non-growing (steady- state)
conditions. The cells raised in the inner portion of zU migrate inward to zFR providing cell to these zones. We therefore concluded that zU is the stem cell zone of the adrenal cortex at least in non-growing rat5.
We recently established adrenocortical cell-lines with
properties of zU cells6 from the adrenal corteces of mice
carrying a temperature-sensitive SV 40T antigen gene. The
cells secreted a hitherto unknown protein identified by subtractive hybridization between the cells without and with
expressing P45011β7. The protein as well as its mRNA was
detected in zG and zU, but not in zFR, and we designated
it adrenocortical zonation factor-1(AZ-1). Its in situ expressions in the adrenocortical zones as well as in Y1 cells
are inversely correlated with the expression of P45011β.
On the basis of these findings obtained by using cytochrome P450 enzymes as markers, mechanisms for the adrenocortical zone formation and its maintenance will be
discussed.
Supported in part by grants-in-aid for General Scientific Research from the Ministry of Education, Science and
Culture, Japan and by grants from Keio University.
WL11 MECHANISM OF THE
FORMATION AND
MAINTENANCE OF THE
FUNCTIONAL ZONATION IN
THE ADRENAL CORTEX
FUMIKO MITANIa, KUNIAKI MUKAIa,
MAKOTO SUEMATSUa, AND YUZURU ISHIMURAb
a
Department of Biochemistry and Integrative Medical Biology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan ,bDepartment
of Biochemistry, University of Texas Health Science Center
at San Antonio, San Antonio, TX 78229-3900, USA,
[email protected]
The adrenal gland consists of two ontogenetically and
functionally distinct endocrine tissues, the cortex and the
medulla. The cortex is mesodermal in origin and derived
from the coelomic epithelium, while the medulla is ectodermal and derived from the neural crest. The cortex is
subdivided further into three concentric zones, the zona
glomerulosa (zG), the zona fasciculate (zF) and the zona
reticularis (zR). These three zones are morphologically and
functionally distinct from each other: zG cells secrete mineralocorticoids, zF cells give glucocorticoids, and zR
cells produce adrenal androgens in many primates. Such
specialization in the function is called „functional zonation“ of the adrenal cortex.
Interestingly, all the enzymes involved in the biosyntheses of these corticosteroids from cholesterol are the members of cytochrome P450 family with an only exception of
3β-hydroxysteroid dehydrogenase/isomerase. The successful identification and purification of aldosterone-synthase
cytochrome P450 (P450aldo) and glucocorticoid-synthesizing cytochrome P450 (P45011β) of rats in early 1990s1, led
us to investigate the molecular basis for such functional zonation of the adrenal cortex as well as mechanisms for the
maintenance and development of rat adrenal glands.
REFERENCES
1. Ogishima T., et al.: J. Biol. Chem. 264, 10935 (1989).
2. Mitani F., et.al.: Endocrinology. 135, 431 (1994).
3. Mitani F., Ishimura Y. Oxygen and Life (ICS 1233).
pp.99. Elsevier Science, Amsterdam 2002.
4. Mitani F., et al.: Endocrinology. 135, 431 (1994).
5. Mitani F., et al.: Biochim. Biophys. Acta. 1619,
317(2003).
6. Mukai K., et al.: Eur. J. Biochem. 269, 1 (2002).
7. Mukai K., et al.: J. Biol. Chem. Accepted for publication on February 24 2003 (readable in the electronic
version).
S64
ration of treatment, dose, organ and species. Thus, to be effective on CYPs, OPZ treatment must be started before or
be concomitant to exposure to the carcinogen. This compound is able to augment CYP1A expression and activity
not only in rat liver but also in kidney, lung and intestine.
Regarding CYP2B1 the transcripts were unchanged in the
lung while the amount of corresponding apoprotein and activity was dramatically decreased, due to a rapid catabolism of the protein by the proteasome degradation pathway9.
As a whole, these results from studies on OPZ demonstrate that evaluation of chemoprotective agents must consider their potential ability to modulate some CYP activities, that these effects can be inhibitory and/or inducing
and suggest that these effects may have considerable consequences on the preventive capacity of such molecules in
vivo.
WL12 DUAL EFFECTS OF THE
CHEMOPROTECTIVE AGENT
OLTIPRAZ ON CYTOCHROMES
P450
SOPHIE LANGOUËT, FABRICE MOREL, and
ANDRÉ GUILLOUZO
INSERM U 456, Université de RENNES 1, 2 avenue du
Professeur L Bernard, 35043 RENNES Cedex, France,
[email protected]
A variety of natural products and synthetic chemicals
are known to have chemopreventive properties; they include dithiolethiones, isothiocyanates, polyphenols, flavonoids and various other antioxidants. They may act by altering the balance between activation and inactivation of
carcinogens through their action on phase 1 and phase 2
xenobiotic metabolizing enzymes. Oltipraz (OPZ), a synthetic derivative of 1,2-dithiole-3-thione, is one of the most
promising chemoprotective agent, based on preclinical studies and a recent phase IIa clinical trial in China1. The chemical was first claimed to protect against carcinogenesis
by inducing enzymes involved in their inactivation, such as
glutathione transferases (GST), NADPH-quinone reductases and UDP-glucuronosyltransferases2. These results obtained from rodent studies were confirmed in humans by
using primary human hepatocytes. However, the GST isoform pattern induced by OPZ was different3.
More recent investigations showed that OPZ also exerted dual effects on cytochromes P450 (CYP). First, we showed that this agent was a rapid and potent inhibitor of
CYP1A2 and CYP3A4 in human hepatocytes4. This observation was confirmed by measuring caffeine N3-demethylation catalyzed by CYP1A2 in OPZ-treated rat liver and
the use of human recombinant CYP1A2 expressed in Escherichia coli membranes5, 6. OPZ acts as a competitive and
mechanism-based inhibitor of CYP1A2 with Ki of 1.5
µM and kinactivation of 0.19 min-1 6. It also inhibits in the following order CYP 3A4, 1A1, 1B1 and 2E1 and one of its metabolites, OPZ M3, had similar effects. This inhibitory effect was confirmed in vivo in humans by OPZ oral administration of 500 mg weekly or 125 mg daily and determination of aflatoxin B1 mercapturic acid formation and caffeine N-demethylation respectively1, 7.
In addition, it was found that after 48hr of OPZ treatment, CYP1A1/2 and CYP2B1/2 were significantly increased in rat liver, at both mRNA, protein and activity levels.
Interestingly, by using differentiated human Caco2 cells,
derived from a colon adenocarcinoma, the induction of
CYPA1 was demonstrated to occur at the transcriptional level and to be mediated by the aryl hydrocarbon receptor
(AhR). An increased intracellular calcium concentration
was required to obtain AhR activation by OPZ8.
From the data of the literature it is obvious that OPZ effects are dependent upon the CYP enzymes, time and du-
REFERENCES
1 Wang, J. S., Shen, X., He, X., Zhu, Y. R., Zhang, B. C.,
Wang, J. B., Qian, G. S., Kuang, S. Y., Zarba, A., Egner, P. A., Jacobson, L. P., Munoz, A., Helzlsouer, K. J.,
Groopman, J. D. and Kensler, T. W. J Natl Cancer Inst
91, 347 (1999).
2 Kensler, T. W., Egner, P. A., Dolan, P. M., Groopman, J.
D. and Roebuck, B. D. Cancer Res 47, 4271 (1987).
3 Morel, F., Fardel, O., Meyer, D. J., Langouet, S., Gilmore, K. S., Meunier, B., Tu, C. P., Kensler, T. W., Ketterer, B. and Guillouzo, A. Cancer Res 53, 231 (1993).
4 Langouët, S., Coles, B., Morel, F., Becquemont, L., Beaune, P., Guengerich, F. P., Ketterer, B. and Guillouzo,
A. Cancer Res 55, 5574 (1995).
5 Langouët, S., Maheo, K., Berthou, F., Morel, F., Lagadic-Gossman, D., Glaise, D., Coles, B., Ketterer, B. and
Guillouzo, A. Carcinogenesis 18, 1343 (1997).
6 Langouët, S., Furge, L. L., Kerriguy, N., Nakamura, K.,
Guillouzo, A. and Guengerich, F. P. Chem Res Toxicol
13, 245 (2000).
7 Sofowora, G. G., Choo, E. F., Mayo, G., Shyr, Y. and
Wilkinson, G. R. Cancer Chemother Pharmacol 47, 505
(2001).
8 Le Ferrec, E., Lagadic-Gossmann, D., Rauch, C., Bardiau, C., Maheo, K., Massiere, F., Le Vee, M., Guillouzo, A. and Morel, F. J Biol Chem 277, 24780(2002).
9 Le Ferrec, E., Ilyin, G., Maheo, K., Bardiau, C., Courtois, A., Guillouzo, A. and Morel, F. Carcinogenesis 22,
49 (2001).
S65
WL13 TRANS-SPECIES
REGENERATING OR
STEM-CELL DERIVED
HEPATOCYTE-LIKE CELLS:
A PERSPECTIVE TO SOLVE THE
AVAILABILITY PROBLEM FOR
HUMAN HEPATOCYTES?
up to 30 000 ‘existing chemicals’ which are currently marketed in volumes greater than 1 ton per year, and for which
essential human health and ecotoxicological data are lacking at a cost, predicted to be several billions. Assuming
no increase in current capacities for chemical testing, the
time scales for testing for chemical risk assessment for human health, proposed, might need to be extended by 36 years for the Base Set Testing, i.e. to 2048.
There is general consensus to develop alternative testing strategies using in vitro test systems, in order to reduce
the time scale for the completion of the testing and to allow
compliance with the new legal demands. This is exactly the
challenging task of the European Centre for the validation
of Alternative Methods (ECVAM) e.g. to make use of the
recent technological advances made with regard to in vitro
toxicological testing in order to validate relevant, reliable
and robust high-thoughput alternative toxicological test
systems targeting specifically the required regulatory toxicological studies for human health.
Approaches for identifying metabolism-mediated effects after chemical exposure and their inclusion in testing
strategies are a high-priority in order to offer alternatives to
in vivo animal testing. The major human CYPs involved in
chemical metabolism are CYP1A1, CYP1A2, CYP2A6,
CYP3A4, CYP1B1, CYP2C9, CYP2C19, CYP2D6, and
CYP2E1. The key phase II enzymes include N-acetyl transferases, UDP-dependent glucuronyl transferases, phenol
sulphotransferases, sulphotransferases, and glutathione
S-transferases. Metabolism is identified as „the bottleneck“ in in vitro test development. So far, no consensus has
been reached on the optimal test systems to be used for obtaining the information required in the human health areas
of high priority such as acute systemic toxicity or reproductive toxicity. However, the most widely-accepted approach is to progress from simple, inexpensive and less-specific in vitro or in silico models, such QSARs and human
liver suspensions and subcellular fractions, to more demanding and more-complex models, such as cultures of
primary human hepatocytes. In this way, the first tier acts
as a preliminary screen for identifying any metabolism-mediated toxic effects followed by more specific tests, such as
the induction of biotransformation enzymes or the occurrence of polymorphism-related effects.
FRANZ OESCH, and JAN-GEORG HENGSTLER
Institute of Toxicology, University of Mainz, Obere Zahlbacher Str. 67, 55131 Mainz/Germany
Hepatocytes represent a valuable tool in the evaluation
of xenobiotics allowing for determination of (i) rate of disappearance of a compound, (ii) metabolic profiles, (iii)
enzyme induction, (iv) drug-drug interaction, (v) cytotoxicity. We have established a technique that allows cryopreservation of hepatocytes in a way that guarantees similar
enzyme activities in cryopreserved versus freshly isolated
hepatocytes of the major drug metabolizing phase I and
phase II pathways and allows examination of enzyme induction in cultures of cryopreserved hepatocytes. Cryopreserved human hepatocytes are available, but rare.
Stimulation of hepatocyte proliferation in vitro is difficult.
We developed a technique that allows proliferation of human
hepatocytes after transplanting into 2/3 hepatectomized nude
rats. With this technique the yield of hepatocytes is rather low.
We therefore sought conditions allowing for differentiation of
human umbilical cord stem cells into the direction of hepatocytes. The cells were transplanted into livers of SCID-mice.
Several hepatocyte markers such as human albumin were successfully brought to expression in these cells, whilst ß2-microglobulin was no longer detectable. This down regulation
promises to represent an escape mechanism from killer T-cells
of the host mice which may constitute an important prerequisite for growing human hepatocyte-like cells in animal hosts.
So far the cells had not yet differentiated into perfect hepatocytes but clearly into hepatocyte-like cells.
WL14 METABOLISM:
A BOTTLE-NECK FOR IN VITRO
REGULATORY TOXICITY TESTS
AND TESTING STRATEGIES
SANDRA COECKE
ECVAM, Institute For health and Consumer Protection,
European Commision Joint Research Centre, Ispra, Italy,
e-mail: sandra.coecke
The recent European Commission (EC) White Paper
‘Strategy for a future chemicals policy’ outlines a plan to
S66
will be a valuable model to study not only the role of hepatic
and extrahepatic P450s in drug disposition, but also the role of
Phase II enzymes and drug transporters.
We have also deleted a Phase II enzyme, glutathione Stransferase Pi (GstP), from mice. GstP has been found to be expressed at elevated levels in a number of animal and human tumours, and in cell lines made resistant to a variety of drugs7,8.
Little, however, is known about the endogenous role of this enzyme. Mice lacking GstP are apparently phenotypically normal, with no change in morbidity or mortality. However, GstP
null mice are found to have significantly higher levels of tumour formation, in the skin or lungs, when challenged chemically9. Furthermore, such mice also have altered sensitivity to
the effects of drugs such as acetaminophen10, involving a mechanism(s) which have not yet been fully elucidated.
WL15 APPLICATIONS OF
TRANSGENIC MODELS
IN DRUG METABOLISM
COLIN J. HENDERSONa, DIANNE CARRIEa,
AILEEN MCLARENa, DIANA OTTOa,
IAN ROSEWELLb, and C. ROLAND WOLFa
a
Cancer Research UK Molecular Pharmacology Unit, Biomedical Research Centre, Ninewells Hospital & Medical
School, Dundee, DD1 9SY, United Kingdom.
[email protected]; bCancer Research UK Transgenic Services, Clare Hall Laboratory, Blanche Lane, South
Mimms, Potters Bar, Herts., EN6 3LD, United Kingdom.
REFERENCES
Transgenic technology has the potential to revolutionise the field of drug metabolism; the ability to manipulate
genetically the expression of individual drug metabolising
enzymes in order to better understand their function(s)
could lead to the development of new drugs for the treatment of diseases such as cancer, as well as the optimisation
of treatment regimes with existing drugs.
Our Laboratory has been interested for several years in
the expression and regulation of drug metabolising enzymes and the role of such enzymes in cellular processes
such as carcinogenesis and drug resistance. Recently, we
have developed several transgenic models to enable us to
study how drug metabolising enzymes protect us from the
chemically challenging environment in which we live.
The cytochrome P450 supergene family represents
a group of Phase I drug metabolising enzymes catalysing the
insertion of an atom of molecular oxygen into a huge range of
diverse substrates1. A number of P450s have been ‘knocked
out’ in the mouse; deletion of those P450s which carry out essentially ‘house-keeping’ reactions tend to result in embryonic lethality, or severe welfare problems, whereas deletion of
P450s which carry out metabolism of xenobiotics tend to display little or no obvious phenotypic changes2,3. This is at least
in part due to the overlapping substrate specificity demonstrated by P450s, and the ability of other isoenzymes to at least
partially compensate for the absence of individual enzymes.
In order to overcome this, we have deleted the sole electron
donor to microsomal cytochrome P450s, cytochrome P450 reductase (CPR); as anticipated, this deletion was embryonic
lethal4,5. However, our targeting strategy incorporated the loxP
sites, allowing us to conditionally delete CPR when used in
conjunction with a mouse line carrying Cre recombinase under the control of a tissue-specific promotor. We have now generated mice which lack hepatic CPR, and thus the P450 system in the liver is essentially inactivated6. Despite this, hepatic CPR null mice grow and develop normally, demonstrating
that the hepatic P450 system is not essential for survival in the
postnatal period, but that; however, they have enlarged, fatty
livers, an almost complete lack of bile production, and a significantly lowered serum lipid profile. Work with these mice
will be presented to demonstrate that hepatic CPR null mice
1. Sligar SG.: Essays Biochem 34, 71 (1999).
2. Buters JT., Doehmer J., Gonzalez FJ.: Drug Metab Rev
31, 437 (1999).
3. Gonzalez FJ., Kimura S.: Arch Biochem Biophys 409,
153 (2003).
4. Otto DME., Henderson CJ., Carrie D., Davey M., Gundersen TE., Blumhoff R., Adams RH., Tickle C., Wolf
CR.: Mol. Cell Biol. submitted (2003).
5. Shen AL., O’Leary KA., Kasper CB.: J Biol Chem 277,
6536 (2002).
6. Henderson CJ., Otto DME., Carrie D., Magnuson MA.,
McLaren AW., Rosewell I., Wolf CR.: J Biol Chem In
Press (2003).
7. Henderson CJ., McLaren AW, Moffat GJ., Bacon EJ.,
Wolf CR.: Chem Biol Interact 111-112, 69 (1998).
8. Hayes JD., Pulford DJ.: Crit Rev Biochem Mol Biol
30, 445 (1995).
9. Henderson CJ., Smith AG., Ure J., Brown K., Bacon EJ,
Wolf CR.: Proc Natl Acad Sci USA 95, 5275 (1998).
10. Henderson CJ., Wolf CR., Kitteringham N., Powell H., Otto
D., Park BK.: Proc Natl Acad Sci USA 97, 12741 (2000).
WL16 CYTOCHROME P450 4FS:
RESPONSE AND ROLE
FOLLOWING BRAIN TRAUMA
HENRY W. STROBELa, AUINASH KALSOTRAa and
PRAMOD K. DASHb
a
Department of Biochemistry and Molecular Biology,
Department of Neurobiology and Anatomy, The University
of Texas-Houston Medical School, P.O. Box 20708, 6431
Fannin Street, 77225, Houston, TX, US
[email protected]
b
Closed head injury resulting from traffic accidents or
other blunt trauma often leads to a severe inflammatory
S67
response in the brain and coma. The inflammatory cascade
mediated in part by leukotriene and prostaglandin signals
results in the migration of cells, ions and fluids into the site
of injury. The production of leukotrienes and prostaglandins is catalyzed by lipoxygenase and cyclooxygenase 2.
respectively, from arachidonic acid. The cytochrome
P4504F subfamily has been implicated by Kikuta and others in metabolizing leukotriene and prostaglandin to products inactive as prompts of the inflammatory cascade and
therefore playing a role in modulating the levels of leukotrienes and prostaglandins present. We report that the levels
of lipoxygenase and cycloxygenase expression reach a maximum of 24 hours after controlled cortical impact in rats
while CYP4F expression is suppresed at this time point. At
later time points CYP4F levels rise while lipoxygenase and
cyclooxygenase levels decrease, marking the recovery
phase of the injury. Similar patterns are observed in the tissues especially in the lung, as the function of time after injury.
hepatocytes. While hepatic CYPs are located primarily in
the ER, in brain, the presence, inducibility and metabolic
activity of many drug metabolizing CYPs have been observed in mitochondrial membranes, in neuronal processes, in
the plasma membrane as well as in the ER.
Induction
Brain CYPs are inducible by many common hepatic inducers (e.g. CYP2B1 by phenobarbital). Many compounds
affect liver and brain differently, e.g. ethanol induces
CYP2B1 in rat liver but not brain, whereas nicotine induces CYP2B1 in rat brain but not liver (for review2). Induction is usually cell-type specific and tends to occur at low
doses. Induction of brain CYPs may be detrimental, for
example by 1) rerouting the metabolism of endogenous
compounds, 2) increasing cellular oxidative stress, and 3)
increasing production of toxic metabolites (e.g. procarcinogens, amphetamine metabolites).
Metabolism by Brain CYPs
CYP enzymatic activity is reported for both rodent and
human brain. Brain membranes have been shown to metabolize the same probe substrates used to assess specific hepatic CYP activity, such as 7-pentoxyresorufin for
CYP2B1/2, 7-ethoxyresorufin for CYP1A1/2, N-nitrosodimethylamine and p-nitrophenol for CYP2E1, dextromethorphan for CYP2D. Many other hepatic CYP substrates
such as bufuralol, amytriptyline, nicotine, phencyclidine,
amphetamines, and neurotoxins such as organophosphorous insecticides are metabolized by brain tissues. While it
is unlikely that brain CYPs contribute to overall clearance
of xenobiotics, their punctate, region- and cell-specific expression suggests that CNS CYP enzyme activity may create micro-environments in the brain with differing drug
and metabolite levels (not detected or predicted by plasma
drug monitoring). Coupled with the sensitivity of CNS
CYPs to induction, this may in part account for interindividual variation in response to centrally acting drugs and
neurotoxins, and may have implications for individual variation in receptor adaptation and cross-tolerance to different drugs.
REFERENCES
1. Cui X., Kalsotra A., Robida A.M., Matzilevich D., Moore A.N., Boehme C.L., Morgan E.T., Dash P.K., Strobel H.W. Biochim. Biophys. Acta. 1619, 325 (2003).
WL17 COMMONLY USED DRUGS
REGULATE CYP ENZYMES
WITHIN THE BRAIN
RACHEL F TYNDALE, LISA HOWARD, KERRI
SCHOEDEL, and SHARON MIKSYS
Centre for Addiction and Mental Health & Dept of Pharmacol, Univ of Toronto, Canada. [email protected]
CYP enzymes in the brain are of interest due to their
potential role in the (in)activation of centrally acting drugs,
in the metabolism of endogenous compounds, and also due
to their ability to generate toxic metabolites and/or oxygen
stress that may cause neuronal damage.
CYP2D6 and Alcoholics
CYP2D6 is expressed in liver, brain and other extrahepatic tissues where it metabolizes a range of centrally acting drugs and toxins. Members of the CYP2D family have
been localized in rat brain where they have a heterogeneous distribution3,4. As ethanol can induce CYP2D in rat
brain, we hypothesized that CYP2D6 expression would be
higher in brains of human alcoholics. We examined regional and cellular expression of CYP2D6 mRNA and protein
by RT-PCR, southern blotting, slot blotting, immunoblotting and immunocytochemistry5. A significant correlation
was found between mean mRNA and CYP2D6 protein levels across 13 human brain regions. Higher expression was
detected in 13 brain regions of alcoholics (n=8) compared
to non-alcoholics (n=5) (ANOVA p<0.001). In hippocam-
Identification and Localization in the Brain
Many CYPs demonstrate constitutive expression within
animal and human brains, are distributed heterogeneously
among brain regions (e.g. members of CYP1, 2, 3 and 4 families), and are found in both neurons and glial cells (for
review1). Many CYPs are located at the blood-brain interface and in circumventricular organs (regions unprotected
by the blood-brain barrier) such as the choroid plexus and
posterior pituitary. Brain CYPs are reported to occur at
about 1% of hepatic levels, but most earlier studies treated
the brain as a homogeneous organ. CYP levels can vary several fold among brain regions, and in specific cells (e.g.
Purkinje neurons) levels can be as high as those found in
S68
pus the higher levels were localized in CA1-3 pyramidal
cells and dentate gyrus granular neurons. In cerebellum
this was localized in Purkinje cells and their dendrites.
Both of these brain regions, and these same cell-types, are
susceptible to damage by alcoholism. A poor metabolizer
(CYP2D6*4/*4) was identified and in this brain sample
there was no detectable CY2D6 protein, confirming the
specificity of the antibody used. This indicates that for
those with genetically deficient variants of the enzyme,
metabolism within the brain will differ from those with the
active forms independently of the presence of inducers. Together these data suggest 1) CYP2D6 in brain is inducible
and 2) that in alcoholics the higher levels of brain CYP2D6
expression may contribute to altered sensitivity to centrally
acting drugs and to the mediation of neurotoxic and behavioral effects of alcohol.
cytes. CYP2B6 levels were higher in brains of smokers and
alcoholics, particularly in cerebellar Purkinje cells and hippocampal pyramidal neurons, cells known to be damaged
in alcoholics. Significantly more (p<0.05) CYP2B6 protein
was seen in 4 brain regions of smoking alcoholics compared with nonsmoking nonalcoholics: hippocampus, caudate
nucleus, putamen and cerebellar hemisphere. The genetic
variant C1459T (R487C) has been associated with reduced
hepatic enzyme levels, stability and activity. Preliminary
genotyping of this small sample (n=24) suggests that individuals with the CC genotype have higher brain CYP2B6
than those with the CT or TT genotype. Higher brain
CYP2B6 activity in smokers and alcoholics may alter the
sensitivity to centrally acting drugs, increase the susceptibility to neurotoxins and carcinogenic xenobiotics, and
may contribute to central tolerance to nicotine.
CYP2E1, Alcohol and Nicotine
Ethanol and nicotine are commonly co-abused drugs.
CYP2E1 metabolizes ethanol and bioactivates tobacco-derived procarcinogens. Ethanol and nicotine can induce hepatic CYP2E16 and we hypothesized that both centrally active drugs could also induce CYP2E1 within the brain7.
Male rats were treated with saline, ethanol (3.0 g kg-1 by
gavage) or nicotine (1.0 mg kg-1 s.c.) for 7 days. Ethanol
treatment significantly increased CYP2E1 in olfactory
bulbs, frontal cortex, hippocampus and cerebellum while
nicotine induced CYP2E1 in olfactory bulbs, frontal cortex, olfactory tubercle, cerebellum and brainstem. Immunocytochemical analysis revealed that the induction was
cell-type specific. Consistent with the increased CYP2E1
found in rat brain following drug treatments, brains from
alcoholics and alcoholic smokers showed greater staining
of granular cells of the dentate gyrus and the pyramidal
cells of CA2 and CA3 hippocampal regions as well as of
cerebellar Purkinje cells compared to nonalcoholic nonsmokers. Moreover, greater CYP2E1 immunoreactivity was
observed in the frontal cortices in the alcoholic smokers in
comparison to nonalcoholic nonsmokers and alcoholic
nonsmokers. To investigate if nicotine could contribute to
the increased CYP2E1 observed in alcoholic smokers, we
treated human neuroblastoma IMR-32 cells in culture and
found significantly higher CYP2E1 immunostaining in nicotine-treated cells (0.1-10 nM). CYP2E1 induction in the
brain, by ethanol or nicotine, may influence the central effects of ethanol and the development of nervous tissue pathologies observed in alcoholics and smokers.
Much remains to be understood before we can firmly
establish relationships between the presence of CYPs in
brain, their function in this highly heterogeneous and complex organ, and the resulting consequences on overall drug
effect and brain function.
Funded in part by the Centre for Addictions and Mental Health, CIHR grant #14173 and a Canadian Research
Chair in Pharmacogenetics to RFT.
REFERENCES
1. Miksys S.L., Tyndale R.F.: Psychiatry Neurosci. 27,
406, 2002.
2. Schoedel K., Howard L., Tyndale R.F: Biochim. Biophys. Acta 1619, 283, 2003.
3. Tyndale R.F, Li Y., Li N-Y., Messina ES., Miksys S.,
Sellers E.M.: Drug Metab. Disp. 27, 924, 1999.
4. Miksys S., Rao Y., Sellers E.M., Kwan M., Mendis D.,
Tyndale R.F: Xenobiotica 30, 547, 2000.
5. Miksys S., Rao Y, Hoffmann E., Sellers E.M., Mash
D.M, Tyndale R.F: J. Neurochem. 82, 1 (2002).
6. Howard L., Micu A., Sellers E.M., Miksys S., Tyndale
R.F: Pharmacol. and Exp. Therap. 299, 542, 2001.
7. Howard L., Miksys S., Hoffmann E., Mash D., Tyndale
R.F: Brit. Pharmacol. (in press).
8. Miksys S., Hoffmann E., Tyndale R.F: Biochem. Pharmacol. 59, 1501, 2000.
9. Schoedel K.A., Sellers E.M., Tyndale R.F: Biochem.
Pharmacol. 62, 1025, 2001.
10.Lerman C., Shields P.G., Wileyto E.P., Audrain J., Pinto
A., Hawk L., Krishnan S., Niaura R., Epstein L.: Pharmacogenetics 12, 627, 2002.
11.Miksys S., Lerman C., Shields P.G., Mash D.C., Tyndale R.F: Neuropharmacology 2003 (in press pending
revisions).
CYP2B6, Alcohol and Nicotine
CYP2B6 metabolizes drugs such as nicotine and bupropion, and many toxins and carcinogens. Nicotine induces CYP2B1 in rat brain8 but not liver9 and in humans polymorphic variation in CYP2B6 affects smoking cessation
rates10. We compared CYP2B6 expression in brains of human smokers and nonsmokers and alcoholics and nonalcoholics (n=26). Human CYP2B6 expression was brain region-specific and was observed in both neurons and astro-
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WL18 THE ARACHIDONIC ACID
MONOOXYGENASES:
P450-EICOSANOIDS AS
ENDOGENOUS REGULATORS
OF ORGAN AND BODY
PHYSIOLOGY
REFERENCES
1. Capdevila J.H., Falck J.R.: Biochem. Biophys. Res.
Commun. 285, 571, and cited references (2001).
2.- McGiff J.C., Quilley J.: Am. J. Physiol. 277, R607, and
cited references (1999).
3.- Imig J.D.: Am. J. Physiol. 279, F965, and cited references (2000).
4.- Holla V., et al.: Proc. Natl. Acad. Sci.(USA) 98, 5211
(2002).
JORGE H. CAPDEVILA, MICHAEL WATERMAN and
VIJAYKUMAR HOLLA
Departments of Biochemistry and Medicine, Vanderbilt
University Medical School, Medical Center North S-3223,
Nashville, TN 37232
WL19 CHIMERIC SIGNALS OF
XENOBIOTIC INDUCIBLE CYPS:
MECHANISM OF
MITOCHONDRIAL TARGETING
AND FUNCTIONAL
IMPLICATIONS
CYP P450s of the 4A and 2C gene subfamilies are
responsible for most of the arachidonic acid epoxidation and
ω/ω-1 hydroxylation catalyzed by human and rodent kidney
microsomal fractions1. The expression of these proteins is
highly regulated and controlled by physiological and pathophysiological factors such as diet, salt intake, hormones, and
diseases such as diabetes and hypertension1,2. By virtue of
their vasoactive and ion transport properties, the epoxyeicosatrienoic and hydroxyeicosatetraenoic acids (EETs and
HETEs, respectively), modulate kidney function and body
fluid and salt homeostasis2,3. Experimental manipulation of
Cyp 4A or 2C gene expression changes the activities of the
renal arachidonic acid epoxygenases and ω/ω-1 hydroxylases
and the kidney biosynthesis of EETs and/or HETEs. These
changes CYP P450-eicosanoid biosynthesis are associated
with alterations in renovascular tone, the capacity of the organ to excrete salt and water, and ultimately, with changes in
systemic blood pressures1-3. Genetically induced alterations
in renal Cyp 4a10 expression and activity, depresses the biosynthesis and urinary excretion of pro-natriuretic, anti-hypertensive EETs, and leads to the development of salt sensitive
hypertension. Targeted disruption of the Cyp 4a14 gene causes the upregulation of the Cyp 4a12 gene, an increase in the
biosynthesis of pro-hypertensive 20-HETE in the kidney, and
renal vasoconstriction4. These Cyp 4a14 (-/-) genotype specific changes in Cyp 4a expression are androgen-mediated
and result in the development of androgen-dependent, sexually dimorphic, hypertension1,4. The presence of androgen-regulated CYP 4A and 2C isoforms in microvessels dissected from rat kidney, suggests that CYP P450 gene transcription and hemodynamic factors are key components of the mechanisms by which the metabolites of the CYP P450 arachidonic acid monooxygenases regulate renal physiology and
systemic blood pressures. Genomic cloning and sequence
analysis shows the existence of two human 4A isoforms,
CYPs 4a11 and 4A22. Only CYP 4A11 is expressed in kidney and catalyzes the hydroxylation of arachidonic acid.
These results, as well as epidemiological evidence suggesting
a role for sex-dependent mechanisms in the pathophysiology
of human hypertension, suggest CYP 4A11 as a novel candidate gene for the analysis of the molecular mechanisms of
human hypertension.
NARAYAN G. AVADHANI
University of Pennsylvania, Philadelphia, PA 19104, U.S.A.
Xenobiotic inducible CYP1A1, 2B1, 2E1 and also
a number of other CYPs contain „atypical“ N-terminal signals at their N-termini, termed „chimeric signals“, and bimodally targeted to both the ER and mitochondria under both
in vitro and in vivo conditions. A positively charged domain
spanning 12-23 amino acids, containing two or more evenly
spaced positive residues, immediately past the N-terminal ER
targeting and transmembrane domains functions as a cryptic
mitochondrial targeting signal in these CYPs. Mitochondrial
targeting of apoproteins appears to be a regulated process requiring the activation of cryptic signal either by endoproteolytic processing at the N-terminus as in the case of CYP1A1
(and also 1A2 and 1B1) or by PKA mediated phosphorylation at internal sites as in 2B1, 2E1 and 2D6. Mitochondrially
targeted CYPs functionally interact with Adx through charge
pairing and catalyze enzymatic activity for different substrates in an Adx+Adr supported system. Under chronic inducer
treatment conditions (BNF) the mitochondrial targeted,
N-terminal truncated CYP1A1 (termed P450MT2) accumulates with time to become a major fraction of cellular/tissue
pool. Furthermore, P450MT2 exhibits high specificity for
N-demethylation of erythromycin, and an array of antidepressants and neuroactive drugs that are not the marker substrates for the intact microsome associated CYP1A1. The
presentation will focus on a) mechanisms of mitochondrial
targeting of CYPs with chimeric signals, b) mode of interaction of mitochondrial targeted CYPs, c) molecular and
biochemical characteristics of the cyotoslic protease, which
is induced/activated by chemical, and or, oxidative stress, and
d) possible physiological and pathological implications of
mitochondrial targeted CYPs.
Supported by NIH grant GM34883-19.
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4
LECTURES – THURSDAY – CL
CL02
REGULATION AND
INTERACTION OF REDOX
PARTNERS WITH
ACTINOMYCETE CYPS
Invited Lectures – CL
CL03
MECHANISM AND
REGULATION OF ELECTRON
TRANSFER IN CYTOCHROME
P450 REDOX SYSTEMS
ANDREW W. MUNROb, OLIVIER ROITELb,
KIRSTY J. MCLEANb, ALDO GUTIERREZb,
JASWIR BASRANb, MARK J.I. PAINEa,
ROBERT FINNa, C. ROLAND WOLFA,
GORDON C.K. ROBERTSb, and NIGEL S.
SCRUTTONb.
DAVID C. LAMBa, LI LEIb, MICHAEL R. WATERMANb
and STEVEN L. KELLYa
a
Wolfson Laboratory of P450 Biodiversity, University of
Wales Aberyswtyth, Aberystwyth, UK and bVanderbilt University School of Medicine, Nashville, Tennessee, USA
a
Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee, DD1 9SY,
UK. bDepartment of Biochemistry, University of Leicester,
University Road, Leicester, LE1 7RH, UK. [email protected]
The genus Streptomyces produces more than two-thirds
of naturally occurring antibiotics and a wide array of other
secondary metabolites. These include numerous antibacterial agents (e.g. erythromycin, tetracycline), anticancer
agents (doxorubicin, adriamycin), immunosuppressants
(FK506, rapamcycin), antiparasitic agents (avermectin, nemadectin), antifungals (amphotericin, griseofulvin, nystatin), cardiovascular agents (lovastatin, compactin) and veterinary products (monensin, tylosine). Invariably, cytochromes P450 are involved in the biosynthetic pathways of
many of these compounds. We report the pattern of mRNA
expression for the cytochrome P450 (CYP)/ferredoxin
(fdx)/ferredoxin reductase (fpr) complement of the model
organism of this genus, Streptomyces coelicolor A3(2).
While transcription for all of the 18 CYPs occurred from
the onset of germination, throughout developmental
growth and secondary metabolism, fdx/fpr expression did
not, indicating that availability of reductase regulated CYP
activity. Exposure of cells for 6h following germination to
the polycyclic aromatic hydrocarbons dimethylbenzanthracene and benzo[a]pyrene, saw a loss of some specific CYP
messages depending on the compound used. However, activation of transcription of a specific fpr gene, FR2
(SCO7117), was observed, indicating that it may interact
with specific CYPs involved in the xenobiotic metabolism
response. As a test of this hypothesis we expressed in
Escherichia coli and purified to homogeneity five CYPs
(CYP105D5, which is specifically involved in xenobiotic
metabolism, and CYP154A1, CYP154C1, CYP158A1 and
CYP158A2, which are putatively involved in secondary
metabolism) and the two ferredoxin reductases (FR2 and
FR3) proposed to shuttle electrons to CYP via iron-sulphur
ferredoxins. Interestingly, FR2 preferentially reduced
CYP105D5 (>90% reduction) compared to the other CYPs
(<20% reduction) while FR3 preferentially reduced
CYP154A1, CYP154C1, CYP158A1 and CYP158A2
(>85%) compared to CYP105D5 (<10%). We propose that
control of CYP activity occurs through expression of specific ferredoxin reductases both during the normal life cycle and following xenobiotic exposure. The results are
extrapolated to other members of actinomycetes.
The cytochromes P450 typically require the delivery of
two electrons from redox partner enzymes to facilitate the
reductive activation of bound molecular oxygen, leading to
the oxygenation of substrate1. In eukaryotic microsomal
systems, these are usually delivered by the diflavin (FADand FMN-containing) enzyme NADPH-cytochrome P450
reductase (CPR)2. CPR-like enzymes are also found in prokaryotes - the most notable example being the fatty acid
hydroxylase cytochrome P450/CPR fusion enzyme flavocytochrome P450 BM3 from Bacillus megaterium3. In recent studies, we have determined the thermodynamic properties of a number of human and bacterial diflavin reductases, including P450 BM3 and human CPR4, 5 (Figure 1).
In both these cases, genetic dissection was used to produce
Fig. 1. Scheme showing the respective reduction potentials for the
FAD and FMN cofactors in various members of the diflavin reductase enzyme family. The midpoint reduction potentials for the
oxidized/semiquinone (ox/sq) and semiquinone/hydroquinone
(sq/hq) transitions of the FAD and FMN cofactors are shown for
flavocytochrome P450 BM3 (BM3), neuronal nitric oxide synthase (NOS), human CPR (CPR) and human novel reductase 1
(NR1). NR1 is the most recently characterized member of the diflavin reductase family14. Potentials are shown relative to that of
the common reductant for these reductases - NADPH (-320 mV).
S71
evolutionary relationship to the ferredoxin NADP + reductases (Figure 2,11). Studies of the „reverse“ (NADP+ reduction)
reaction
with
dithionite-reduced
human
FAD/NADPH domain indicate that the hydride transfer reaction occurs more rapidly in this direction (~ 8 s-1) than in
the physiologically relevant direction (~ 3 s-1) under the experimental conditions9. This is in accordance both with the
proposed evolutionary origin of the domain, and with the
respective reduction potentials for the NADP+/NADPH and
human CPR FADH2/FAD redox couples (-320 mV and 333 mV, respectively,5).
the separate FAD/NADPH-binding and FMN-binding domains of the enzymes, enabling the kinetic and potentiometric properties of the individual flavins to be studied in
isolation. Thermodynamic properties of the isolated flavin
domains were found to be identical, within error, to those
for the intact CPR enzymes. These data support early theories of the evolutionary origin of CPR-like enzymes from
fusions of genes encoding ferredoxin NADP+-reductaselike and flavodoxin-like progenitors6, and are also in agreement with structural data for the rat CPR isoform, which
shows the flavin-binding domains to be clearly spatially
separated7. Our recent kinetic studies on the BM3 and human CPR enzymes have revealed novel regulatory features; specifically as regards the mechanisms by which control is exerted over the processes of electron transfer between NADPH and FAD, and between FAD and FMN cofactors.
Stopped-flow kinetic studies of human CPR reveal the
importance of Trp-676 in the control of the reductive reaction by NADPH8. In rat CPR, The aromatic ring of Trp-677
shields the isoalloxazine ring of the FAD flavin from solvent. The corresponding Trp-676 in human CPR should
have the same structural configuration, and studies on
W676H and W676A mutants confirm the importance of the
residue in human CPR8. Trp-676 acts as a „trigger“ to release NADP+, and release of NADP+ from the active site of
the W676H mutant, post hydride transfer to the FAD,
was retarded. This led to an accumulation of a stable
EH2-NADP+ complex in stopped-flow mixing experiments
with NADPH, due to slow dissociation of NADP+. Analysis of the concentration dependence of FAD reduction in
human CPR and its FAD/NADPH domain revealed that
electron transfer was accelerated at low [NADPH], suggesting the presence of a second, inhibitory NADPH-binding
site in the enzyme8, 9. The inhibitory site has lower affinity
for NADPH, but its population results in retardation of release of NADP+ from the catalytic site - and persistence of
the charge transfer complex. We have recently observed
a similar phenomenon in the adrenodoxin reductase homologue FprA from Mycobacterium tuberculosis (see accompanying paper from McLean et al.,10). In FprA there is a remarkable (up to 8-fold) acceleration in the NADPH-toFAD electron transfer observed as [NADPH] is decreased
below 100 µM.
The P450 BM3 CPR and FAD/NADPH domains did
not exhibit this pattern of accelerated NADPH-to-FAD
electron transfer at low [NADPH], instead showing a more
„normal“ positive, hyperbolic dependence of electron transfer rate on [NADPH] (see accompanying paper from Roitel et al.,11). However, studies of the ability of the 4-electron reduced CPR domain and 2-electron reduced
FAD/NADPH domains to pass electrons to NADP+ indicated that the rate of this reaction was reciprocally related to
[NADP+]. For P450 BM3 we envisage that the dual pyridine nucleotide binding site model remains viable, but that
for this enzyme there may be discrimination of the second
binding site towards NADP+, perhaps reflecting a closer
Fig. 2. Reaction scheme for interaction of the CPR domain of
P450 BM3 with oxidized and reduced nicotinamide nucleotide cofactors. Oxidized (E, Eox) and reduced (EH2) forms of the enzyme
interact with either NADPH or NADP+ cofactors, which may bind
to either a catalytic (stronger) or non-catalytic (weaker) pyridine
nucleotide binding site. Occupation of the non-catalytic site is indicated by the appearance of the NADP+ in parentheses (although
NADPH can also occupy the second site). The EH2NADP+ and
EoxNADPH species are in dynamic equilibrium, and the reaction
is reversible - yielding either EH2 and NADP+ (following dissociation of NADP+ from reduced enzyme) or Eox and NADPH (following dissociation of NADPH from oxidized enzyme). Both equilibrium species may bind a second molecule of NADP+(H) to the
second (non-catalytic site), and NADPH can also bind NADPH to
the catalytic site after population of the non-catalytic site by
NADP+(H).
To investigate the rates of internal (i.e. between FAD
and FMN cofactors) electron transfer in human CPR, temperature jump relaxation spectroscopy was used. In key experiments, the enzyme was pre-reduced to a 2-electron level under anaerobic conditions using either dithionite or
NADPH as the reductant. Following a rapid (4 µs) temperature jump (of approximately 7 oC), the rate of electronic
re-equilibration in the systems was determined. In preceding potentiometric studies, the proximity of the FAD oxidised/semiquinone and FMN semiquinone/hydroquinone
was established, suggesting that thermal perturbation of the
2-electron reduced enzyme would affect the proportions of
enzyme existing in either FAD semiquinone/FMN semiquinone or FAD oxidised/FMN hydroquinone forms5. This
proved to be the case, and a rate of 55 s-1 was determined
for inter-flavin electron transfer in the NADPH-reduced
enzyme12. The corresponding rate for dithionite-reduced
enzyme was only 11 s-1, demonstrating that nicotinamide
coenzyme binding is of considerable importance in regulating the internal electron transfer rate. Increasing solution
viscosity dramatically reduced inter-flavin electron transfer rate, suggesting conformational gating of the process 12.
More recent studies have examined the influence of the
S72
binding of molecular „fragments“ of pyridine nucleotide
cofactors on the electron transfer reaction in human CPR 13.
Collectively, these data have provided new insights into the
mechanisms of control of reactivity in the diflavin reductase enzyme family.
cation, multiduplication or amplification of active genes, most
likely in response to dietary components that have resulted in
a selection of alleles with multiple non-inducible genes. An
updated list of variant CYP alleles is present at the Home
Page of the Human Cytochrome P450 (CYP) Allele Nomenclature Committee (http://www.imm.ki.se/CYPalleles/). Several examples exist where subjects carrying certain alleles suffer from a lack of drug efficacy due to ultrarapid
metabolism or, alternatively, adverse effects from the drug
treatment due to the presence of defective alleles. Dosage
requirements for several commonly used drugs that have
a narrow therapeutic range can differ more than 20-fold
dependent on the genotype or the enzyme expression status. By contrast, carcinogen metabolising cytochrome
P450s are less polymorphic and no firm relationships have
been established linking increased risk for cancer with any
specific P450 polymorphism. In the present overview recent aspects of cytochrome P450 polymorphism is discussed. A new cellular level for the polymorphic expression
of functional cytochrome P450s of different specificities,
namely trans-splicing, is presented. Furthermore, new polymorphic CYP alleles will be presented which affect the
ability for induction of gene expression.
REFERENCES
1. Munro A.W., Lindsay J.G.: J. Biol. Chem. 266, 13469
(1991).
3. Miles C.S., Ost, T.W.B., Noble M.A., Munro A.W.,
Chapman S.K.: Biochim. Biophys. Acta 1543, 383
(2000).
4. Daff S., Chapman S.K., Turner K.L., Holt R.A., Govindaraj S., Poulos T.L., Munro A.W:. Biochemistry 36,
13816 (1997).
5. Munro A.W., Noble M.A., Robledo L., Daff S., Chapman S.K.: Biochemistry 40, 1956 (2001).
6. Porter T.D.: Trends Biochem. Sci. 16, 154 (1991).
7. Wang M., Roberts D.L., Paschke R., Shea T.M., Masters B.S.S., Kim J.-J.P.: Proc. Natl. Acad. Sci. USA 94,
8411 (1997)
8. Gutierrez A., Doehr O., Paine M., Wolf C.R., Scrutton
N.S., Roberts G.C.K.: Biochemistry 39, 15990 (2000).
9. Gutierrez A., Lian L.-Y., Wolf C.R., Scrutton N.S., Roberts G.C.K.: Biochemistry 40, 1964 (2001).
10.McLean K.J., Scrutton N.S., Munro A.W.: Biochem. J.
(in press) (2003).
11. Roitel O., Scrutton N.S., Munro A.W.: Biochemistry
(submitted).
12.Gutierrez A., Paine M., Wolf C.R., Scrutton N.S., Roberts G.C.K.: Biochemistry 41, 4626 (2002).
13.Gutierrez A., Munro A.W., Grunau A., Wolf C.R., Scrutton N.S., Roberts G.C.K.: Eur. J. Biochem. (submitted).
14.Finn R.D., Basran J., Roitel O., Wolf C.R., Munro
A.W., Paine M.J.I., Scrutton, N.S.: Eur. J. Biochem. (in
press) (2003).
CL04
CL05
ROLE OF GENETIC
POLYMORPHISM, DRUG
EXPOSURE AND SEX FOR
EXPRESSION AND FUNCTION
OF HEPATIC CYTOCHROMES
P450
ULRICH M. ZANGER
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstr. 112, D-70376 Stuttgart, Germany;
[email protected]
About one dozen P450 isozymes of families CYP1,
CYP2 and CYP3 are collectively responsible for most
phase I biotransformations in human liver. Variability in
the expression and function of these isozymes is a major
cause for unpredictable drug response. Principal mechanisms underlying inter- and intraindividual variability are
genetic polymorphisms, regulation of expression by xenobiotic and endogenous substances, as well as physiological
factors including sex, liver disease and others. To dissect
the contributions of these different factors to P450 variability, we established a large human liver bank consisting of
over 300 surgical tissue samples with extensive clinical documentation. Recent work concentrated on families 2 and
3, including CYPs 2D6, 2B6, and the 3A family.
CYP2D6 is involved in the metabolism of about one
fourth of all drugs. Its expression is highly polymorphic
with more than 70 known alleles which code for protein
MECHANISMS AND
CONSEQUENCES OF
INTERINDIVIDUAL VARIATION
IN CYTOCHROME P450
FUNCTIONAL GENOMICS
MAGNUS INGELMAN-SUNDBERG
Division of Molecular Toxicology, IMM, Karolinska Institutet, SE 171 77 Stockholm, Sweden
The majority of human P450-dependent xenobiotic metabolism is carried out by polymorphic enzymes which can
cause abolished, quantitatively or qualitatively altered or enhanced metabolism. The latter situation is due to stable dupli-
S73
products with variant or lacking function1. This leads to
four phenotypes in the Caucasian population which are termed ultrarapid (UM), extensive (EM), intermediate (IM)
and poor metabolizers (PM) depending on their drug oxidation capacity. Using liver samples of phenotyped patients, we have shown that these phenotypes are the result of
different protein expression levels with UM, EM, IM, and
PM expressing about 24, 8, 2.5 and 0 pmoles of CYP2D6
per mg of microsomal protein2. In about 20% of UMs, increased expression is known to be due to the presence of three or more gene copies (e.g., 2D6*2x2). The residual 80%
of UMs are genetically less well predictable because the involved genotypes are also frequent among EMs. By contrast, the IM phenotype is a distinct subgroup of about 1015% of the population most of whom have the genotype
*IM / *PM, with *IM designating a functionally impaired
allele (*41, *9, *10) and *PM designating any nullallele 3.
Recent work on the most frequent *41 allele, which was
previously shown to be genetically distinct from *2 in a putative NFkappaB binding site at -1584 bp3 suggests that this
promoter polymorphism can not explain the large difference
in phenotypic expression between *2 and *41. Mutation
scanning revealed a novel intronic mutation that was found
to be specific for *41. RT-PCR experiments on liver RNA
from genotyped indicviduals demonstrated a close correspondence between its presence and a nonfunctional 2D6
splice variant. Together, these data provide molecular explanations for the differentiated genetic determination of
CYP2D6 function, which has been known for over 25 years.
The CYP3A isozymes 3A4, 3A5, 3A7 and 3A43 are expressed at very different levels in human liver with
CYP3A4, the major form4, contributing critically to the metabolism of at least half of all drugs. Although a number of
polymorphic CYP3A4 alleles were recently identified 1,
these are rare and do not make a significant contribution to
CYP3A4 expression variability, which was found to be 50fold both at the level of protein (6 to 295 pmoles per mg of
microsomal protein) and ~70-fold at the level of mRNA.
More important is inducibility by endogenous and exogenous substances which bind as ligands to the orphan nuclear
receptors PXR (pregnane X receptor) or CAR (constitutive
androstane receptor), thereby causing up-regulation of gene
transcription. By analyzing CYP3A4 expression in human
liver samples in relation to pre-surgical drug exposure, it
was observed that treatment with carbamazepine or St.
John’s wort lead to an approx. four-fold in vivo induction of
3A4-protein, but most drugs produced smaller or no effects.
Because there is substantial evidence for sex-related differences in drug pharmacokinetics in humans, in particular
for CYP3A4 substrates, we systematically analyzed a larger
number of liver samples. By carefully controlling for confounding factors, we observed for the first time a sexual dimorphism in CYP3A4 expression with women expressing
roughly twice the amount of 3A4 protein than men. In contrast to CYP3A4, expression of both 3A5 and 3A7 was
found to be largely determined by genetic polymorphisms.
Human CYP2B6 recently gained more attention since it
was shown to be involved in the metabolic activation and
inactivation of a number of clinically important drugs, including cyclophosphamide and bupropion. CYP2B6 is inducible by a range of substances including phenobarbital, and
the nuclear receptors CAR and PXR play a role in the induction process. In the first systematic polymorphism analysis of the CYP2B6 gene, we could show that 2B6 is
highly polymorphic with numerous alleles coding for variant proteins with different expression levels5. For example,
a mutation in exon 9 (R487C) was associated with almost
8-fold decreased protein levels in human liver. Furthermore, we recently identified more than ten novel alleles
with amino acid changes, some of which affect expression
and function drastically, as shown by expression in insect
cells. Thus, with respect to the balance between genetic
and nongenetic factors controlling expression, CYP2B6
appears to take an intermediate position between CYP2D6
and CYP3A4.
These studies demonstrate the value of large human liver collections to dissect the various factors responsible for
variability in P450 expression and function and they contribute to the understanding of interindividual variability in
drug response.
Supported by the Ministry of Education and Science,
Deutsche Forschungsgemeinschaft, and the Robert Bosch
Foundation Stuttgart.
REFERENCES
1. The CYPallele nomenclature homepage:
http://imm.ki.se/CYPalleles/
2. Zanger UM, Fischer J, Raimundo R, Stüven T, Evert
BO, Schwab M, Eichelbaum M.: Pharmacogenetics 11,
573, (2001)
3. Raimundo S, Fischer J, Eichelbaum M, Griese E-U,
Schwab M, Zanger UM.: Pharmacogenetics 10, 577,
(2000)
4. Koch I, Weil R, Wolbold R, Brockmöller J, Hustert E,
Burk O, Nuessler A, Neuhaus P, Eichelbaum M, Zanger
UM, Wojnowski L.: Drug Metabolism and Disposition
30, 1108, (2002).
5. Lang T, Klein K, Fischer J, Nüssler AK, Neuhaus P,
Hofmann U, Eichelbaum M, Schwab M, Zanger UM.:
Pharmacogenetics 11, 399, (2001).
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CL06
ated with these transitions come from crystallographic data
on P450cam. The substrate-free low-spin enzyme is six-coordinated with a water covalently bound to iron at L6 which
is buried in the active site cavity. The substrate camphor
binds to the protein moiety (will be referred to as Site 1),
releasing the L6 water converting the heme to five-coordinated high-spin state. The product 5-exo-OHCamphor (P)
binds to Site 1 as well as to the iron (Site 2) through its OH group forming Fe-O bond resulting in six-coordinated
low-spin complex. Whereas five-coordinated Site 1 complexes are high spin the Site 2 complexes are low spin.
Since the heme is buried in the active site cavity, the Site 2
would not be directly accessible to exogenous P therefore
the P is not expected to bind to both sites at the same time.
We investigated this by transient difference spectroscopy
using P4502B4, by the Temperature-jump relaxation technique. The static difference spectrum produced by the substrate benzphetamine (S) was normal Type I difference
spectrum. The static spectrum produced by the product
des-methyl benzphetamine (P) which forms low-spin complex was much smaller and distorted with little absorption
increase at 390nm. However in the time-scale of the Tjump technique, the transient difference spectrum produced by the P was also normal Type I spectrum with the expected absorption increase at 390nm, indicative of highspin complex,. This indicates that low-spin EP complex
formation may proceed via. transient high-spin intermediate. It is possible that the time difference between product
binding to Site 1 and Site 2 is such that appreciable amount
of high spin EP complex could be observed in the T-jump
time scale. This indicates that product binding to form low
spin complex may be a two-step process.
ENVIRONMENTAL GENOMICS
ON PLANT P450 SPECIES
METABOLIZING HERBICIDES
HIDEO OHKAWA
Kobe University, Research Center for Environmental
Genomics, Kobe, Japan, [email protected]
Current genome projects revealed that there are 3 P450
genes Saccharomyces cerevisiae, 57 genes in Homo sapiens, 80 genes Caenorhabditis elegans, 88 genes in Drosophila melanogaster, approximately 150 genes in Phanerochaete chrysosporium, 273 genes in Arabidopsis thaliana and 458 genes in Oryza sativa.
Particularly, higher plants were found to have outstandingly large number of P450 genes. However, the enzyme
function is known for around 60 P450 species in plants. We
worked on systematic reverse genetics of the P450 superfamily in Arabidopsis. Based on the combination of cytochrome P450 databases, T-DNA mutant lines of Arabidopsis, in planta transformation of Arabidopsis and heterologous expression systems, the function of CYP71B11 and
CYP86A1 was identified. CYP71B11 catalyzed hydroxylation of sulphonylurea herbicides and seemed to be involved
in the herbicide selectivity and resistance. CYP86A1 catalyzed w-hydroxylation of fatty acids, and involved in production of cutin monomers. This P450 species also seemed
to be related to tolerance to the herbicide pyributicarb.
Discussions include application of P450 species metabolizing herbicides for phytoremediation and monitoring
of environmental contaminants in plants.
CL07
TRANSIENT DIFFERENCE
SPECTROSCOPY OF
SUBSTRATE (S) AND PRODUCT
(P) BINDING TO CYP450:
(A NOVEL INSIGHT INTO
PRODUCT BINDING)
SHAKUNTHALA NARASIMHULU, and
JOHN K. WILLCOX
University of Pennsylvania, HDSR, Philadelphia,
Pa. 19104,
Substrate-induced low- to high-spin state transition reflected as shift in the Soret band from ∼417nm to ∼390nm
has been observed with many P450s (E). This is the basis
for the well known S-induced Type I difference spectrum
with absorption minimum at ∼420nm and maximum at
∼390nm and an isosbestic point at ∼406nm, indicative of
high spin complex. Insights into structural changes associ-
S75
03 PostMP.QXD 10.6.2003 12:06 Stránka 76
Poster Presentations – MP
5
POSTERS – MONDAY – MP
the terphenyl rings against the porphyrin plane leading to
comparatively more rigid pocket on the other side for improved selectivity. Similarly, enantioselectivity is also
improved by pyridine bases with catalyst 2. In this case,
the electronic effect may play a major role. We have carried out epoxidation of cis-β-methylstyrene using 2 and pyridines of various electronic properties. A linear free
energy relationship has been established for electron donating region. And the best enantioselectiivty is 81 % ee.
MP01 AXIAL LIGATION FOR
ENHANCING SELECTIVITY IN
CATALYTIC EPOXIDATION BY
CYTOCHROME P450 MODELS
CHI. K. CHANG, TAT-SHING LAI, and
LAM-LUNG YEUNG
REFERENCES
Departement of Chemistry, Hong Kong University of
Science & Technology, Clear Water Bay, Hong Kong,
[email protected]
1. Suslick K.S. in: Comprehensive Supramolecular Chemistry (Lehn J.M., ed.), vol. 5, 141, Elsevier, London
1996.
2. Collman J.P., Zhang X., Lee V.J., Uffelman E.S., Brauman J.L: Science 261, 1404 (1993).
3. Lai T.S., Ng K.H., Liu H.Y., Chang C.K., Yeung L.L.:
Synlett 1475, (2002).
4. Lai T.S., Lee S.K.S., Yeung L.L., Liu H.Y., Williams
I.D., Chang C.K.: J. Chem. Soc. Chem. Commun. 5,
620, (2003).
Shape selectivity of cytochrome P450 monooxygenase
to a large extent comes from the narrow hydrophobic
pocket formed by the arrangement of amino acid residues
pointing toward the oxidation reaction site. To mimic such
a pocket, heme model compouds have been developed to
demonstrate shape selectivity in the oxidation of hydrocarbon substrates.1,2 Typically, such compounds are difficult to
synthesize and the selectivity is not particularly remarkable. The catalytic activity and selectivity of metalloporphyrins in biomimetic oxidation chemistry of cytochrome
P450 can be affected by the axial coordination. We have
observed a remarkable axial ligand effect for enhancing regioselectivity in diene epoxidation by a barrel-shaped
porphyrin 1 as well as for altering enantionselectivity in
methylstyrene epoxidation by a chiral porphyrin 2.3, 4
MP02 FUNCTIONAL ANALYSIS OF
CYP2D6.31 - ARG440HIS
SUBSTITUTION DIMINISHES
ENZYME ACTIVITY BY
DISRUPTING REDOX PARTNER
INTERACTION
DELPHINE ALLORGEa, DIDIER BREANTa,
JACKY HARLOWb, JOEY CHOWDRYb,
JEAN-MARC LO-GUIDICEa, DANY CHEVALIERa,
CHRISTELLE CAUFFIEZa, MICHEL LHERMITTEa,
FRANK E. BLANEYc, GEOFFREY T. TUCKERb,
FRANCK BROLYa and SOLO WYNNE ELLISb
a
EA2679, Faculté de Médecine/Pôle Recherche, 1 place de
Verdun, 59045 Lille, France, [email protected], bUniversity of Sheffield, Unit of Molecular Pharmacology and
Pharmacogenetics, Division of Clinical Sciences, Royal
Hallamshire Hospital, Sheffield S10 2JF, United Kingdom,
c
GlaxoSmithKline Research and Development, New Frontiers Science Park (North), Third Avenue, Harlow CM19
5AW, UK.
Using metal-oxo species derived from manganese
porphyrin 1 with PhIO or MCPBA, enhancements in regioselectivity towards less substituted C=C bond in diene
epoxidations can be achieved by simply adding organic bases as axial ligand. For example, the selectivity to produce
8,9-limoene oxide can be elevated from 33% (ligand free)
to 66% (with DMAP or 4-t-butylpyridine). We have examined the effect of various bases and ruled out the electronic effect to be solely responsible for this phenomenon.
A plausible explanation is the presence of structural modulation that the binding of organic bases in the highly crowded environment would restrict the rotational freedom of
Cytochrome P450 2D6 is an important human drug-metabolizing enzyme that exhibits a marked genetic polymorphism. Numerous inactivating mutations of the
CYP2D6 gene have been characterized and homozygous or
compound heterozygous individuals for such non-functional alleles exhibit a poor metabolizer phenotype. However,
the consequences for expression and/or catalytic activity of
a substantial number of other allelic variants of CYP2D6
S76
ked heme, demonstrating that E310 is required for covalent
binding. All three mutants show decreased rates of lauric
acid hydroxylation with the A310 and G310 mutants exhibiting altered regioselectivity favoring ω-1 and ω -2 hydroxylation of lauric acid respectively. Uniquely, the aciddissociable heme obtained from the D310 mutant exhibited
the same chromatographic behavior as a hydroxymethylheme species released upon base treatment of the covalently linked wild-type enzyme. Expression of D310 in
H218O enriched media resulted in incorporation of the heavy isotope into the hydroxymethylheme at a molar ratio of
~0.8:1. Finally, the CYP4B1 hydroxymethylheme released
from the wild-type enzyme upon base treatment was further fractionated to detect isomeric products. Comparison
of HPLC UV/Vis traces of these hemes with hydroxymethylheme standards suggests a mixture of 5-hydroxymethylheme (85%) and 8-hydroxymethylheme (15%). These data
(i) show that E310 serves as the site of covalent attachment
of the heme to the protein backbone of rabbit CYP4B1; (ii)
demonstrate the mechanism of covalent heme attachment
most likely involves a heme carbocation species; and (iii)
suggest that heme is bound to wild-type CYP4B1 in two
distinct orientations.
This work was supported by GM49054 and GM07750.
that occur at very low frequencies remain to be determined.
One such rare allele, CYP2D6*31, is characterized by mutations encoding three amino-acid substitutions: Arg296Cys,
Arg440His and Ser486Thr. The identification of this allele in
an individual with an in vivo PM phenotype prompted us to
analyze the functional consequence of these substitutions
on enzyme activity using yeast as a heterologous expression system.
We demonstrated that the Arg440His substitution, alone
or in combination with Arg296Cys and/or Ser486Thr, altered
the kinetics of debrisoquine 4-hydroxylation and dextromethorphan O-demethylation such that their intrinsic clearances (Vmax/Km) were decreased by over 95% compared
to those observed with the wild-type enzyme. The rate of
oxidation of rac-metoprolol at single substrate concentrations was also markedly decreased with each mutant form of
protein containing the Arg440His substitution. These in vitro
data confirm that the CYP2D6*31 allele encodes essentially a non-functional enzyme compatible with an in vivo
PM phenotype, and that the Arg440His exchange is the inactivating mutation. A homology model of CYP2D6 based
on the crystal structure of rabbit CYP2C5 locates Arg440 on
the proximal surface of the protein. Docking the structure
of the FMN domain of human cytochrome P450 reductase
to the CYP2D6 model suggests that Arg440 is a key member
of a cluster of basic amino acid residues important for binding between the two proteins and efficient electron transfer.
MP04 α-NAPHTHOFLAVONE AS
A MODEL LIGAND OF THE
ACTIVE SITE OF CYP3A
MP03 COVALENT HEME BINDING TO
CYP4B1 VIA GLU310 AND
A CARBOCATION PORPHYRIN
INTERMEDIATE
LUCIE BO¤EK-DOHALSKÁ, PETR HODEK and
MARIE STIBOROVÁ
Department of Biochemistry, Faculty of Science, Charles
University, Albertov 2030, 128 40 Prague 2, Czech Republic
[email protected], [email protected]
BRIAN R. BAERa, YI-MIN ZHENGa,
MATTHEW J. CHEESMANa, NICOLE
MOGUILEVSKYb, KENT L. KUNZEa, and
ALLAN E. RETTIEa
Determination of the structure of the binding sites of
the CYP protein molecules and the structure-function relationships are in the center of interest. Understanding this
area may increase our knowledge useful for development
of new drugs as well as for decreasing activation of many
carcinogens. From this point of view, the CYPs of
a 3A subfamily are the most intensively studied. The activities of human CYP3A4 are regulated by binding of various ligands, which leads either to their stimulation or inhibition. The flavonoid α-naphthoflavone (α-NF) is one of
such compounds (ligands). It is often employed as a selective inhibitor of CYP1A1/2. However, α-NF interacts also
with other CYP enzymes, human CYP3A4 and rabbit
CYP3A6, influencing their activities. We determined stimulation of 17β-estradiol 2-hydroxylation catalyzed by
CYP3A, while other CYP3A activities tested in our study
were inhibited; the inhibition of erythromycin and tamoxifen N-demethylation was reversible, while progesterone
and testosterone 6β-hydroxylation was inhibited by α-NF
a
Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195, [email protected],
b
Applied Genetics, Faculty of Science, Universite Libre de
Bruxelles, Gosselies, Belgium
Cytochrome P450 4B1 (CYP4B1) is a principally extrahepatic P450 isoform that is found in mammalian lung
and kidney tissues. Recent investigations have shown that
CYP4B1, and other members of the CYP4 family, are covalently linked to their prosthetic heme group through an
ester linkage with the protein. Sequence alignments of covalently linked CYP4 enzymes indicate a unique glutamic
acid in the I-helix motif FEGHDT as the site of attachment.
This residue was mutated to glycine, alanine, and aspartate
to evaluate the role of E310 in covalent heme binding and
catalysis. None of these mutants displayed covalently lin-
S77
in an irreversible manner1. These results suggest that the
effect of α-NF on CYP3A activities is strongly dependent
on individual substrate. Moreover, here we show that stimulation and/or inhibition effects of α-NF are dictated by
the complexity of the CYP3A enzymatic system. In the enzymatic systems, where oxidation of CYP3A substrates is
very effective (microsomes and Supersomes™), α-NF irreversibly inhibited testosterone 6β-hydroxylation. In other
systems (CYP3A reconstituted with NADPH: CYP reductase) the CYP3A activity is stimulated or not affected. The
results obtained in our studies may be utilized for suggestion of a model for the protein domain of the CYP3A
active site. Our results indicate that CYP3A contains at
least two, or probably three, distinct binding sites for substrates. We found that α-NF is reversibly bound to the same
binding site as erythromycin and tamoxifen, competing
with them for binding to the active center of CYP3A. The
second binding site, distinct from the first one, is the binding site for progesterone and testosterone. To this site,
α-NF reactive species, generated by its oxidation with
CYP3A, and causing the irreversible inactivation of the enzyme, are expected to be bound. Evidence for the third substrate-binding site arises from analysis of 17β-estradiol
2-hydroxylation in the presence of α-NF, cooperativity of
α-NF binding to CYP3A and from the mode of the protection caused by 17β-estradiol against α-NF-mediated
CYP3A inactivation.
Supported by the Ministry of Education of the Czech
Republic (grant MSM 1131 00001) and the Grant Agency
of the Czech Republic (grant 523/01/0840).
The aim of the present study was to evaluate the respective contribution of these residues to the catalytic efficiency and to the regio- and the strereospecificity of the
CYP 2C11. We constructed single or double mutants, coexpressed them with NADPH cytochrome P450 reductase
in baculovirus/insect cell system and determined the testosterone metabolism in microsomal fractions. P450
spectra from all the mutants were similar to that of wild
type microsomal fractions.
Each of nine substitutions in the wild type allele diminished the catalytic activity without modifications in the
regio- and the stereospecificity. None of ten subtitutions in
the Gunn allele was able to restore the level of the wild
type protein.
These results will be discussed by comparison with the
X-ray crystal structure published for CYP 2C5 (1) which
exhibits close similarity with CYP 2C11.
REFERENCE
1. Williams P.A., Cosme J., Shridar V., Johnson E.F.,
McRee D.E.: Mol. Cell, 5, 121 (2000)
MP06 CAMPHOR HYDROXYLATION
BY CYTOCHROME P450CAM:
A THEORETICAL STUDY BY
HYBRID QUANTUM
MECHANICAL/ MOLECULAR
MECHANICAL (QM/MM)
METHOD
REFERENCES
1. Bofiek-Dohalská L., Hodek P., ·ulc M., Stiborová M.:
Chem.-Biol. Interact. 138, 85 (2001).
SHIMRIT COHENa, SASON SHAIKa,
JAN C. SCHÖNEBOOMb, HAI LINb, and
WALTER THIELb
MP05 STRUCTURE-FUNCTION
RELATIONSHIP IN
CYTOCHROME P450 CYP 2C11
a
Department of Organic Chemistry and the Lise MeitnerMinerva Center for Computational Quantum Chemistry,
The Hebrew University of Jerusalem, 91904 Jerusalem, Israel, [email protected], bMax-Planck-Institut für
Kohlenforschung, Mülheim an der Ruhr, Germany
C. CELIERa, C. BIAGINIb, R.C. MAROUN,c and
R. PHILPOTb
Cytochrome P450cam catalyzes the stereoselective and
regioselective hydroxylation of camphor, which produce,
with high efficiency, 5-exo-hydroxycamphor. The active
species of P450cam, CpdI, is an oxo-iron-portoporphyrin
complex, which is ligated to a cysteinato amino acid. Our
recent theoretical study on CpdI1 in the enzyme environment revealed that it behaves as a chameleon and adapts its
electronic character and geometry to the environment. Due
to the significant effect of the environment on CpdI, we
examined the mechanism for camphor hydroxylation by
CpdI within the native protein environment. We used hybrid QM/MM calculations with UB3LYP density functional
QM Hamiltonian. In these calculations, a suitably trunca-
a
INSERM U473, 84 rue du general Leclerc, 94276 Le
Kremlin-Bicetre cedex, France, bNIEHS-LST, PO Box
12233, MD F204, Research Triangle Park, NC 27709,
USA, cUnite de Bioinformatique Structurale, Institut Pasteur, 25-28 rue du Dr Roux, 75724 Paris cedex, France
Among cytochrome P450 family, CYP 2C11 (isolated
from male rat liver) is characterized by its ability to hydroxylate testosterone at 2 positions : 2-alpha and 16-alpha. We
have previously shown that the decrease in testosterone hydroxylating activity observed in an allelic variant (Gunn rat) is
associated with 3 mutations (aminoacids 1, 116 and 187).
S78
ted CpdI species is treated with the QM method, while the
protein environment is treated with MM. The results show
that the reaction proceeds in a stepwise manner via the rebound mechanism and exhibit a two-state reactivity scenario in agreement with gas-phase calculations2,3. The mechanistic and energetic features of the hydroxylation reaction as well as a comparison to the gas phase results will be
presented4.
rease in the heme pocket hydration in 1PB-3A4 complex.
Moreover, increasing hydrostatic pressure results in a marked increase in the cooperativity of 3A4. In our interpretation these findings reveal a conformational transition,
which results from binding of the first molecule of ligand
(1PB) and is central to the allosteric mechanism. This transition apparently decreases the accessibility of the active
site so that water flux into the heme pocket is impeded and
the high-spin state of the heme iron is stabilized. This hypothesis is also supported by the results of stop-flow and
pressure-jump experiments on the kinetics of 3A4 interactions with 1PB and BCT. The unusual stability of the highspin state of 3A4 complex with 1-PB was observed only in
3A4 oligomers. Upon dissociation of 3A4 oligomers in the
presence of detergents the difference in barotropic properties between 3A4-1PB and 3A4-BCT complexes disappears, suggesting involvement of subunit interactions in the
allosteric mechanism. (This research was supported by
grant GM54995 from the NIH).
References:
1. Schöneboom J.C., Lin H., Reuter N., Thiel W., Cohen
S., Ogliaro F., Shaik S., J. Am. Chem. Soc., 124, 8142
(2002).
2. Ogliaro F., Harris N., Cohen S., Filatov M.,. de Visser
S. P, Shaik S., J. Am. Chem. Soc., 122, 8977 (2000).
3. Kamachi T., Yoshizawa K., J. Am. Chem. Soc. ASAP.
4. Cohen S., Shaik S., Schöneboom J.C., Lin H., Thiel W.
In preparation
MP07 CYTOCHROME P450 3A4
ALLOSTERIC MECHANISM
STUDIED BY HIGH-PRESSURE
SPECTROSCOPY
MP08 A CHARGE-PAIRING BUNDLE
AROUND CYS-154 IS PIVOTAL
FOR THE ALLOSTERIC
MECHANISM IN CYTOCHROME
P450ERYF
DMITRI R. DAVYDOV, and JAMES R. HALPERT.
DMITRI R. DAVYDOV,
ALEXANDRA BOCHKAREVA, SANTOSH KUMAR,
and JAMES R. HALPERT.
Department of Pharmacology and Toxicology, The University of Texas Medical Branch, Galveston, TX
[email protected].
Department of Pharmacology and Toxicology,
The University of Texas Medical Branch, Galveston, TX,
[email protected].
Hydrostatic pressure perturbation was used to study the
allosteric mechanisms in human microsomal cytochrome
P450 3A4 (3A4). This approach is especially valuable for
studies of P450-substrate interactions due to the central
role of heme pocket hydration in the mechanism of the substrate-induced spin shift in the P450 heme iron. The pressure-related behavior of 3A4 with the non-allosteric substrate bromocriptine (BCT, Kd = 0.3 µM) was quite similar
to that reported earlier for other cytochromes P450. A pressure induced-spin shift in both substrate-free (∆Vo =
-29 ml/mol) and substrate bound (∆Vo = -40 ml/mol)
enzyme was detected at rather low pressures, so that the
percentage of high-spin 3A4 was negligible above 2.5 kbar.
At higher pressures this process was accompanied
by a P450➝ P420 transition (∆Vo = -36 ml/mol, P1/2 =
2.7 kbar) that involves about 70% of the total hemoprotein.
However, the behavior of 3A4 with the allosteric substrate
1-pyrene butanol (1PB, S50 = 12 µM, n = 1.6) was in drastic contrast to that described above. In this case the pressure-induced high ➝ low spin shift was observed only in
substrate-free enzyme. At high concentrations of 1PB the
amplitude of the spin shift was very low, showing that hydrostatic pressure induces no substrate dissociation nor inc-
In order to explore the allosteric mechanism in cytochrome P450eryF we studied the effect of chemical modification and site-directed mutagenesis of the unique surface-exposed cysteine residue (Cys-154). Binding of 1-pyrenebutanol (1PB) to P450eryF exhibits distinct positive
cooperativity (S50 = 13 µM, n = 2.6). The modification of
Cys-154 with 7-diethylamino-3-(4’-maleimidylphenyl)-4methylcoumarin (CPM) results in complete disappearance
of the allosteric phenomenon. The same result was observed when Cys-154 was replaced with isoleucine. We also
found that an increase in ionic strength suppresses the
cooperativity of the enzyme. The ionic strength dependence of the Hill coefficient exhibits two distinct minima
at I = 0.25 M (n = 1.2) and I = 0.85 M (n = 1.5). Importantly, this decrease in cooperativity is associated with
a profound shift of the spin equilibrium of the substratebound enzyme towards the high-spin state. CPM modification of Cys-154 as well as the C154I mutation completely
prevents these effects.
Cys-154 is located in the loop between D and E α-helices and is surrounded by a network of salt bridges formed
S79
by amino acid residues located in close proximity. We hypothesized that the binding of the first substrate molecule
to the enzyme promotes the dissociation or rearrangement
of these salt bridges that modulate the spin equilibrium in
the enzyme-substrate complex. Modification of Cys-154 as
well as its replacement with isoleucine impairs this mechanism and deprives the enzyme of its allosteric properties.
These results indicate that the rearrangement of a chargepairing bundle between the C, D and E α-helices in
P450eryF is crucial for allostery and substrate-induced
spin transitions in the enzyme. (This research was supported in part by grant H-1458 from the Robert A. Welch
Foundation and grant GM54995 from the NIH).
Current results further demonstrate the utility of molecular models to explain differences in substrate selectivity
between P450 1A1, 1A2 and their mutants. For example,
the preference of P450 1A2 for methoxyresorufin and P450
1A1 for ethoxyresorufin may be rationalized by substrate
dynamics within the active site. In P450 1A2 WT, methoxyresorufin binds with the methoxy group close to the ferryl oxygen and remains in its proximity during molecular
dynamics. On the other hand, this substrate is highly mobile in P450 1A1 WT and the site of oxidation is much farther from the ferryl oxygen. This hinders methoxyresorufinO-dealkylation, consistent with the experimental data. In
contrast, ethoxyresorufin, which is an excellent substrate
for P450 1A1 WT, binds with its ethoxy site of oxidation in
constant proximity of the ferryl oxygen. Similar reasoning
can be applied to explain changes of activity in mutant
P450s. For example, the loss of methoxyresorufin-O-dealkylase and ethoxyresorufin-O-dealkylase activities in P450
1A2 L382A may be explained by the findings that sites of
oxidation for both substrates did not approach the ferryl
oxygen in simulations and substrate mobility was greatly
increased. In general, the loss of activity observed in several P450 1A1 and 1A2 mutants may be linked to increased
substrate mobility and binding orientations unfavorable to
substrate oxidation.
Supported by NIH grants CA85542 and RR16440.
MP09 MOLECULAR MODELING OF
ALKOXYRESORUFIN
OXIDATION BY P450 1A1, 1A2
AND THEIR MUTANTS
SPENCER S. ERICKSEN,
and GRAZYNA D. SZKLARZ
Department of Basic Pharmaceutical Sciences, School of
Pharmacy, West Virginia University, Morgantown, WV 26506,
[email protected]
REFERENCES
The two members of P450 1A subfamily, 1A1 and 1A2,
share 72% amino acid sequence identity, but display different substrate specificities and inhibitor susceptibilities.
Some substrates are metabolized by both enzymes, but
with different efficiencies. For example, 1A1 effectively
oxidizes 7-ethoxyresorufin, while 1A2, although able to
metabolize this substrate, exhibits preference towards the
7-methoxyresorufin. Substrate specificity, product profiles,
and product formation rates may be attributed to a substrate’s tendency to satisfy the catalytic requirement of site
approach to the activated oxoheme. The substrate must assume an appropriate near attack configuration for the reaction to proceed and to yield product. Therefore, the replacement of residues present in the active site should alter
substrate binding orientation and dynamics, and thus product formation rates and the extent of uncoupling.
The homology models of P450 1A11 and 1A2 were
used to dock enzyme substrates into the active site and to
identify key residues that might be important for activity.
Several of these key residues are different between the two
enzymes. Thus, we have constructed several reciprocal active site mutants of 1A1 and 1A2, evaluated their methoxyresorufin-O-dealkylase and ethoxyresorufin-O-dealkylase activities, and compared the results with those obtained from docking and molecular dynamics simulations of
the substrates bound in models of P450 1A1 and 1A2 wild
type and mutants. These methods have been successfully
used to explain the effect of mutations at position 382 in
P450 1A12.
1. Szklarz G.D., Paulsen M.D., J. Biomol. Struct. Dyn. 20,
155 (2002).
2. Liu et al., 2003, Drug Metab. Disp., in press.
MP10 CAN P450 BM3 TURN OVER
SUBSTRATES TYPICALLY
METABOLISED BY CLASS II
HUMAN P450S?
EVIDENCE ON THE
FUNCTIONAL RELATIONSHIP
OF P450 BM3 WITH THE
HUMAN ENZYMES
ANDREA FANTUZZI, and GIANFRANCO GILARDI
Department of Biological Sciences, Imperial College
London, SW7 2AZ, [email protected]
Present in the three domains of life, cytochrome P450
monooxygenases constitute one of the most diverse enzyme superfamilies. In most cases, the cytochrome P450
redox partners are synthesised as separate polypeptides.
However, in the fatty acid hydroxylase P450 BM3 of Bacillus megaterium, the haem domain is fused at the carboxyl terminus with a NADPH:cytochrome P450 reductase
S80
module. As self-sufficient enzyme, it has the highest catalytic activity known for a P450 monooxygenase. The molecular phylogeny illustrates that the BM3 cluster, with
a eukaryotic (fungal) and prokaryotic branch, is more related to eukaryotic P450s than to most of the prokaryotic
P450s. By virtue of its similarity to microsomal P450s this
soluble prokaryotic enzyme has served as a 3D model for
the membrane-associated eukaryotic P450s. Inspired by
the domain structure of P450 BM3, several soluble fusion
proteins comprising class II P450s fused with NADPH:cytochrome P450 reductase have been expressed to facilitate
their biochemical characterisation.
Can P450 BM3 turn over substrates typically metabolised by classII human P450s? To address this question 16
drugs, clustered in 4 different groups depending on the specificity for different human P450s, were screened using
a high-throughput screening method1. 7 positives were
identified and further characterised. The Km determined
for all the drugs investigated is in the millimolar range,
1000 fold higher that the one determined for the human
P450s. Nevertheless due to the high catalytic efficiency of
P450 BM3, the observed Vmax is 10 to 100 times higher
than the values reported in literature for the human enzymes. The metabolic products were analysed and characterised by HPLC.
These findings suggest that P450 BM3 can be used not
only to derive 3D models for human P450s, but also as an
experimental model for the human enzymes where in
a common scaffold few alterations account for different
functional characteristics.
contribution of other active site residues to substrate binding and orientation. We have recently described a structural model of CYP2D6 that identifies the aromatic residue
Phe120 (located in the B’-C loop) as a likely major feature
of the active site topology, aiding substrate binding. To
examine the role of Phe120, substitutions to Ala, Leu, Tyr,
Ser and His have been prepared in bacterial membranes coexpressing the human cytochrome P450 reductase. Subsequent characterization has been carried out using the prototypical CYP2D6 activities bufuralol 1’ hydroxylation
and dextromethorphan O- and N-demethylation. A peak representing a novel hydroxylated dextromethorphan metabolite was observed in the HPLC trace for the Phe120 Ala
mutant, indicating a role for the aromatic side chain in substrate orientation. The larger effects on Km values determined for dextromethorphan O-demethylation compared
with those for bufuralol 1’ hydroxylation, indicate a substrate dependent role in binding for Phe120. The results
presented here indicate that the aromatic character of the
Phe120 side chain is important in aiding dextromethorphan
binding, while it seems to have a non-specific space filling
role in bufuralol binding.
MP12 RESONANCE RAMAN SPECTRA
REVEAL SUBTLE DIFFERENCES
IN HEME STRUCTURE OF P450
ENZYMES
J. HUDEâEKa, P. ANZENBACHERb, T. SHIMIZUc,
A.W. MUNROd, and T. KITAGAWAe
REFERENCES
1. Tsotsou G.E., Cass A.E.G., Gilardi G.: Biosensors and
Bioelectronics, 17, 133 (2002).
MP11
a
Dept. Biochem. Charles Univ., Albertov 2030, 12840
Praha 2, Czech Rep. [email protected], bInst. Pharmacol, Fac. Med., Palacky Univ., 77 515 Olomouc, Czech
Rep., cTohoku Univ., Inst. Multidisciplinary Res. Adv. Mat.,
Katahira, Sendai,980-8577 Japan, dDept. Biochem., Univ.
Leicester, LE1 7RH UK, eCent. Integrat. Biosci., Okazaki
Natl. Res. Inst., 444 Okazaki, Japan
THE ROLE OF PHE120 IN
CYP2D6 SUBSTRATE BINDING
FLANAGAN, J.U.a, KEMP, C.b, SUTCLIFFE,
M.b, PAINE, M.J.I.a, ROBERTS, G.C.K.c,
and WOLF C.R.a,d
P450 enzymes are able to metabolize a wide variety of
substrates. Obviously, the main responsibility for this variability lies with their apoprotein part, on the other hand,
a question might arise, how much are the differences between various P450 isoforms (and P450-type proteins, such
as NO synthase oxygenase domain) reflected in the structure of their active prosthetic group itself and in the interactions of the heme with the apoprotein part of the molecule.
Resonance Raman spectroscopy offers a possibility to
study the structural details of the heme moiety and therefore might bring some insight into this question. Recently,
we reported differences in heme vinyl orientation of various P450s, as judged from the vinyl stretching vibrations at
around 1620 cm-1.
a
Biomedical Research Centre, University of Dundee, Dundee DD1 9SY, UK, [email protected], bDepartment
of Biochemistry and Department of Chemistry, University of
Leicester, Leicester LE1 7RH, UK, cBiological NMR Centre
and Department of Biochemistry, University of Leicester,
P.O. Box 138, Leicester LE1 9HN, UK, dCancer Research
UK Molecular Pharmacology Unit, Biomedical Research
Centre, University of Dundee, Dundee DD1 9SY, UK.
Although residues influencing CYP2D6 substrate specificity with respect to basic nitrogen containing compounds are well characterised, little is known about the
S81
Tab. 1: Comparison of low-frequency resonance Raman spectra of P450 proteins and
model hemeproteins (positions of Raman bands in cm-1, assignments according to5,6)
Protein
ν8
δ(CβCcCd)
CYP3A4
342
376
420
TW
CYP1A2
346
377
421
TW
CYP51
348
377
CYP2B4
347
380
CYP101
347
379
CYP102
344
380
δ(CβCaCb) + δ(CβMe)
Ref.
418
TW
(405)a
423
[1]
424
[2]
393 (397)b
423
TW ([3])
CYP119
345
380
390, 400
(430)
[4]
CYP11A1
344
379
391, 399
(415)a, 420
[1]
NOSoxy
349
380
390, 405
430
TW
Mb Fe(III)
344
376
409
440
[5]
413, 421
[6]
Cyt c (II)
347
376,382
c
394 , 401
c
a
band not apparent in the measured spectra, found by a peak-fitting procedure
holoenzyme [3]
c
δ(CβCaS)
b
REFERENCES
1. Hildebrandt P., Heibel G., Anzenbacher P., Lanhe R., Krüger V., Stier A.:
Biochemistry 33, 12920 (1994).
2. Wells A.V., Li P., Champion P. M.,
Martinis S. A., Sligar S. G.: Biochemistry 31, 4384 (1992).
3. Deng T., Proniewicz L.M., Kincaid
J.R., Yeom H., MacDonald I. D.G.,
Sligar S. G.: Biochemistry 38, 13699
(1999).
4. Koo L.S., Tschirret-Guth R.A., Straub
W. E., Moënne-Locoz P., Loehr T.M.,
Ortiz de Montellano P.R.: J. Biol.
Chem. 275, 14112 (2000).
5. Hu S., Smith K.M., Spiro T.G.:
J.Am.Chem.Soc. 118, 12638 (1996).
6. Hu S, Morris I.K., Singh J.P., Smith
K.M., Spiro T.G.: J.Am.Chem.Soc.
115, 12446 (1993).
MP13 FLUORESCENCE CORRELATION
SPECTROSCOPY STUDY OF
INTERACTION BETWEEN
CYP 3A4 AND COUMARIN 6
Unfortunately, these vinyl modes are to some extent
obscured by the presence of strong skeletal vibration of
porphyrin (ν10) in the same spectral region. In this work,
we therefore examined the vinyl modes in low-frequency
spectral region several P450 proteins. (CYP 102 A1 from
B. megaterium, both the flavocytochrome and isolated
heme domain and several its mutants, namely F393H,
Y51F and W96Y, CYP51 from Mycobacterium tuberculosis, and human recombinant CYP 1A2 and CYP 3A4 in
presence and absence of substrate/inhibitor and in resting
and reduced state. For a comparison, the heme (oxygenase)
domain of neuronal nitric oxide synthase (nNOS) was also
studied.
In the low frequency region, the pattern of vinyl bending modes is different between the P450 proteins studied
so far. (Tab. 1) At least two different groups might be distinguished: the first one does not have any observable
band between the propionate bending (at around 380 cm -1)
and the vinyl/methyl bending at approximately 420 cm-1
(CYP 3A4, 1A2, 51, 2B4, 101). The second group (CYP
102, 119, 11A1 and the heme domain of nNOS) has a doublet located around 390/400 cm-1 (or a single broad band
above 400 cm-1). This variability may reflect either a difference between vinyl substituents in 2- and 4-position, or
some restriction in vinyl mobility, as suggested by the
comparison with spectra of cytochrome c, where the corresponding groups are covalently linked to the apoprotein
via thioether bonds.
M. BENE·a,b, J. PEKÁREKa,b, M. HOFb,
P. ANZENBACHERc, and J. HUDEâEKa
a
Dept. Biochem. Charles Univ., Albertov 2030, 12840
Praha 2, Czech Rep. [email protected], bJ. Heyerovsk˘ Inst. Phys. Chem AV âR, Dolej‰kova 3, 182 23 Praha
8, Czech Rep., cInst. Pharmacol., Palack˘ Univ., Hnûvotínská 3, 77 515 Olomouc, Czech Rep.
Fluorescence correlation spectroscopy is a method capable to provide information about diffusion constants of
fluorescent compounds in solution. We used this method to
investigate the binding of coumarin 6 (3-(2-benzothiazoyl)-7-N,N-diethylaminocoumarin), resorufin, 7-O-ethylresorufin and hypericin to the human recombinant cytochrome P-450 3A4. From these compounds, the coumarin
6 is the best suitable bright fluorescent dye; the other compounds studied behave in a similar way to coumarin 6, but
the data are more difficult to analyze.
Titration of coumarin 6 solution in buffer with cytochrome
P-450 solution resulted in formation of a fluorescent complex
coumarin-enzyme, manifested by appearance of a slower diffusing component (diffusion time τ = 0.64 ms) in addition to
that of free coumarin (τ = 31 µs). This diffusion time is larger
than expected for a spherical molecule of the size of CYP 3A4
(which should be about 0.15 ms), suggesting a significant deviation of the molecular shape from sphericity.
Acknowledgment
J. Hudeãek and P. Anzenbacher thank for financial support of the Grant Agency of Czech Republic (GACR
203/02/1512).
S82
At concentrations of cytochrome P-450 exceeding 1
complex to 424nm in the Soret band and to 540nm in the
µmol/l, the diffusion time of the slowly diffusing compo-
α-β region and blue-shifted the spectra of reduced complex
nent (representing the enzyme-coumarin complex) remarkably increased to about 1.1 ms. Most probably, this increase reflects an aggregation of P-450 molecule at concentrations higher than this limit. The size of aggregate might
only be estimated (as it depends on the shape of the particle); most probably these are hexamers. (As reported previously for CYP 2B6 and 101 []).
The aggregation apparently prevents full binding of the
fluorescent molecule (coumarin 6), which is demonstrated
by the persistence of the fast diffusing component at even
high P450 concentrations.
Whereas the increase of buffer ionic strength (up to 300
mM) did not change this aggregation behavior, addition of
ethanol to final concentration 200 mM (< 1%) prevented
the formation of aggregates and allowed protein titration to
almost full coumarin 6 binding.
at 400MPa to a new spectra centered at 420nm in the Soret
with a splitting of the α-β bands centered at 558nm (higher
peak) and at 526nm (lower peak). Such presure-induced
spectra transitions correspond respectively to the effect of
pressure on the oxidized P450cam to yield the oxidized inactivated P420 and on the reduced P450cam (Soret band at
412nm) to yield a reduced P420 in which the Soret band is
red-shifted to 427nm with a huge increase in the extinction
coefficient and the α-β bands are splitted into a higher peak
at 560nm and a lower peak at 530nm. Similar results are
obtained by using the other large ligands and inhibitors.
Compression of the active site of P450cam results in an expulsion of larger ligands and possibly an alteration of the
substrate access channel in a way to prevent their rebinding. In addition the heme iron of the pressure-induced reduced cytochrome P420 is hexacoordinated the spectra of
which ressembles that of reduced cytochrome b5 or cytochrome c. Such results are discussed in term of a high pressure-induced huge alteration of the active site capable of
bringing close to the heme iron an histidine residue forming a sixth ligand. However the strenght of binding
and/or the dynamics of such potential sixth ligand are weak
enough to allow the binding of carbon monoxide and cyanide to the heme iron.
Acknowledgment
J. Hudeãek and P. Anzenbacher thank for financial support of the Grant Agency of Czech Republic (GACR
203/02/1512).
MP14 PROBING THE ALTERATIONS IN
THE ACTIVE SITE OF P420CAM:
HIGH PRESSURE STUDIES ON
THE P450CAM-LIGANDS AND
-INHIBITORS COMPLEXES
MP15 CYP ENZYMES:
MOVING TOWARDS VIRTUAL
SCREENING
G. HUI BON HOA
NATHALIE JOURDANa, and ANNAH MANCYb
INSERM U473, Le Kremlin Bicêtre cedex, France
[email protected]
a
Research Technology department, bPharmacokinetics, Dynamics and Metabolism department, Pfizer Global Research (Development, Fresnes Laboratories, 3- 9 rue de la
Loge, 94265 Fresnes Cedex, France.
We have shown that cytochrome P450cam is a relatively compressible protein (pressure-induced shift of the
Soret band: Jung, C., Hui Bon Hoa, G., Davydov, G.D.,
Gill, E., Heremans, K., Eur. J. Biochem. 233, 600 (1995))
and that pressure as high as 250-300 MPa, leads to a deformation of the heme pocket (pressure-induced deformation of the cytochrome P450cam active site: R.A., Tschirret-Guth, G., Hui Bon Hoa, and P.R. Ortiz de Montellano,
J. Am. Chem. Soc. 120, 3590 (1998)). To get more insight
into the structural alterations of the active site, we has undertaken the study of the effect of pressure on the complexes between different states of the heme iron with various
exogenous ligands and inhibitors such as CN, imidazole,
4-phenyl-imidazole and metyrapone. Starting with metyrapone bound to the oxidized P450cam (Soret band at 420nm
and two α-β bands at 568 and 536nm) and to the reduced
P450cam (Soret band at 442nm and two α-β bands at 539
and 564nm plus one smaller peak at 524nm), increase pressure till 300 MPa red-shifted the spectra of the oxidized
Cytochrome P450 is the major enzyme system responsible for catalysing the oxidation of xenobiotics. Therefore,
CYP enzymes do play a very important role in clearance
and drug interactions. In silico models were built from a set
of chemically diverse molecules from literature or in house
data either on human liver microsomes or on recombinant
human CYP assays. Those models are able to indicate if
a new molecule will be a substrate or not of a CYP enzyme
and it requires only the 3D structure of the molecule. Therefore, it identifies the physicochemical and structural features which drive the affinity for the CYP enzyme. This
method can be used at a screening level to take into
account an ADME property and to guide chemical synthesis.
For the CYP2C9 and CYP2D6 enzymes, the data were
respectively divided into a training set of 65 and 70 molecules to build the QSPR model and a testing set of 30 and
S83
40 compounds to validate the models. The descriptors used
come from the VOLSURF program which produce molecular descriptors from GRID molecular Interaction Fields
(MIF). The MIF are obtained with four probes: DRY probe
which simulates hydrophobic interactions, N1 amide nitrogen probe which simulate H-bond donor interaction,
O carbonyl oxygen probe simulating H-bond acceptor interaction and a positively charged probe.
For the CYP3A4 enzyme the data were divided into
a training set of 90 molecules and a testing set of 40 molecules. Five probes are used: H2O, DRY, O, N:= and an
amphiphilic probe.
Partial Least Square analysis was applied and the final
models have 3 PLS components with r2training = 0.74, Q2training
= 0.65 and r2testing = 0.69 for CYP2C9, r2training = 0.8, Q2training
= 0.77 and r2testing = 0.72 for CYP2D6 and r2training = 0.71,
Q2training = 0.61 and r2testing = 0.65 for CYP3A4.
conclusion is only apparent and can be explained by the
dual role of heme pocket water molecules. Water molecules can both make polar interactions to the CO ligand, inducing a red-shift of the CO stretch mode, and compensate
electrostatic potentials of the protein on the distal side, leading to the loss of originally existing contacts to the CO
ligand which results in a blue shift of the CO stretch mode.
With this model our data indicate that an increased heme
pocket hydration should disturb ligand-distal side contacts
which are needed for a specific proton transfer in oxygen
activation resulting in an increased uncoupling H 2O2 formation.
Funding: Deutsche Forschungsgemeinschaft (Ju229/1-1;
Ju229/3-1; SK35/3-3,4); European Commission (BIO2942060).
MP17 INTERMEDIATE RADICAL
FORMATION BY THE
IRON-OXO COMPLEX
PRODUCED IN THE REACTION
OF CYTOCHROME P450CAM
WITH OXIDANTS
MP16 HEME POCKET
COMPRESSIBILITY IN
P450CAM-CO: HIGH PRESSURE
FTIR STUDIES ON THE CO
LIGAND STRETCH VIBRATION
C. JUNG , B. CANNY , J.C. CHERVIN , and
G. HUI BON HOAc
C. JUNGa, V. SCHÜNEMANNb, F. LENDZIANc,
J. CONTZENa, A.-L. BARRAd, and
A. X. TRAUTWEINb
a
a
a
b
b
Max-Delbrück-Center for Molecular Medicine D-13125
Berlin, Germany; bUniversité Pierre et Marie Curie, Laboratoire de Physique des Milieux Condensés, F-75252 Paris, France, cINSERM U473, Le Kremlin Bicêtre cedex,
France, [email protected]
Max-Delbrück-Center for Molecular Medicine, D-13125
Berlin, Germany, bInstitute of Physics, Medical University
Lübeck, D-23538 Lübeck, Germany, cMax-Volmer Laboratory for Biophysical Chemistry, Technical University Berlin, PC 14, D-10623 Berlin, Germany, dGrenoble High
Magnetic Field Laboratory, CNRS, B.P. 166, F-38042 Grenoble Cedex, France, [email protected]
The effect of hydrostatic pressure on the stretch vibration band of the CO ligand in P450cam-CO bound with various substrates is measured with a membrane driven sapphire anvil high-pressure cell mounted in the Fourier transform infrared spectrometer (FTIR). The in-situ pressure in
the cell is determined with the ruby R1 luminescence band.
For different substrate complexes the frequency of the CO
stretch vibration is linearily shifted to lower values with
increasing pressure. The slope of the pressure-induced shift
is interpreted as isothermal compressibility of the heme
pocket and found to be related to the high-spin state content which is produced by the different substrates in the
Fe(III)-form of the enzyme. The compressibilities determined by FTIR would suggest that the P450-substrate complex with 100% high-spin state content is more compressible than the substrate-free complex which is in contradiction to the previously reported results obtained using the
visible absorption spectroscopy (pressure-induced shift of
the Soret band: Jung, C., Hui Bon Hoa, Davydov, G.D.,
Gill, E. , Heremans, K.: Eur. J. Biochem. 233, 600-606
(1995)). It will be demonstrated that such contradictory
The hydroxylating key intermediate in the reaction
cycle of P450 is generally assumed to be a ferryl iron
(Fe(IV); SFe=1) and a porphyrin-π-cation radical (S=1/2)
(compound I) in analogy to chloroperoxidase. For P450,
however, this electron configuration has not unequivocally
been proven experimentally. We have recently found for
wild type P450cam (CYP101) that a freeze-quenched intermediate (8ms - 200ms) in the reaction of peroxy acetic
acid with the substrate-free enzyme is characterized by
a ferryl iron (Fe(IV); SFe=1) and a radical (Srad=1/2) which
is not located at the porphyrin. By high field EPR at 94
GHz and 285 GHz we have now unambiguously identified
the radical site as a tyrosyl radical. The simulation of the
hyperfine structure of the 94 GHz EPR spectrum of wild
type P450cam yields the orientation of the tyrosyl radical
side chain. When assuming no major structural change induced by radical generation as compared with the X-ray
structure, the obtained data are compatible only with Y96.
In mutant Y96F the 9.6 GHz as well as the 94 GHz EPR
S84
spectra show significantly different hyperfine structures as
compared with those of the wild type. The deduced tyrosyl
side chain orientation agrees now perfectly with Y75. No
significant radical signal could be detected in the
Y96F/Y75F double mutant excluding the formation of tyrosine radicals but also of a porphyrin radical. For
P450cam with tyrosines closely located to the heme we
suggest that an initially formed, but up to now undetected,
porphyrin cation radical is reductively quenched by intramolecular electron transfer from the tyrosines.
Funding: Deutsche Forschungsgemeinschaft (Ju229/41,2; TR97/26-1,2); European Community (Programme
„Access to Research Infrastructure Action of the Improving Human Potential Programme „, Grenoble High Magnetic Field Laboratory, Experiments No. SO4001 and
SO4302. We thank S. G. Sligar for providing the plasmid
carrying the Y96F gene.
bilities for low level expressible CYPs as well. The SERR
spectrum of CYP102 heme domain (0.2 mM) shows the
same features as reported previously on milliliter scale 1.
Clearly the ferric state marker band is shown (ν4 at 1372
cm-1), several bands are found in the 1600 cm-1 region, as
previously reported, and also there is a shoulder of the ν29
vibration at 1400 cm-1. Addition of lauric acid to CYP102
before aggregation with the silver particles induced several
changes in the SERR spectrum in the 1100 cm-1 region and
also in the 1500 cm-1 region where the increase in the ratio
of ν29 /ν4 is pronouncedly found. With the current setup, the
SERRS technique can be applied to study the CYP102
heme domain on a picomole scale, giving good spectral information with high resolution and seems promising for
studying human CYPs as well.
REFERENCES
1. Macdonald, I., Munro, A. and Smith, W., 1998: Biophysical J., 74, 3421.
MP18 MICROLITER SCALE SURFACE
ENHANCED RESONANCE
RAMAN SCATTERING ON
CYTOCHROME P450 BM3
MP19 MEMBRANE TOPOLOGY OF
P45017α STUDIED BY
CHEMICAL MODIFICATIONS
AND MASS-SPECTROMETRY
PETER H.J. KEIZERSa, ALOIS BONIFACIOb,
JAN N. M. COMMANDEURa,
GERT VAN DER ZWANb, CEES GOOIJERb,
SASKIA M. VAN DER VIESc, and
NICO P.E. VERMEULENa
HIROSHI KANEKOa, SHUNSUKE IZUMIb,
TAKESHI YAMAZAKIa, TOSHIFUMI HIRATAb, and
SHIRO KOMINAMIa
a
LACDR/Division of Molecular Toxicology, Department of
Pharmacochemistry, bDepartment of Analytical Chemistry
and Applied Spectroscopy, cDepartment of Biochemistry
and Molecular Biology, Vrije Universiteit, De Boelelaan
1083, 1081 HV Amsterdam, The Netherlands
e-mail: [email protected]
a
Faculty of Integrated Arts and Sciences and bGraduate
School of Science, Hiroshima University, 7-1-7 Kagamiyama, Higashihiroshima 739-8521, Japan,
[email protected]
P45017α locates on the membranes of endoplasmic
reticulum of adrenal and gonadal glands and catalyzes
17α-hydroxylation and C17-20 bond cleavage reactions of
C21-steroids. Since hydrophobic substrate steroids are
concentrated in the membranes, it is a reasonable proposal
that the substrate entrance in the enzyme is oriented towards the membrane and the substrate accesses to the binding site directly from the membrane. P450 accepts electrons from NADPH-P450-reductase through the interaction
with the reductase on the membrane. The membranes play
important roles in the reaction of P450s and the membrane
topology of P450 has been investigated by various methods. We have developed a new method for investigation
of the topology by combination of chemical modifications
and mass-spectrometry.
Recombinant guinea pig P45017α (His)4 was purified
and was incorporated into liposome membranes by the cholate dialysis method using egg york phosphatidylcholine.
Acetylation of lysine residues of P45017α was carried out
with acetic anhydride at pH 9. Glycinamidation of acidic
Cytochrome P450s play a major role in the oxidative
metabolism of a large variety of lipophilic endogenous and
exogenous compounds. These enzymes contain a heme domain, needed to activate molecular oxygen that can subsequently be incorporated in the substrate. Cytochrome P450
BM3 (CYP102), from the prokaryote Bacillus Megaterium,
is a cytosolic P450 that is often used as a model for the
membrane bound human CYPs. Surface Enhanced Resonance Raman Scattering (SERRS) is a powerful tool to explore the active site of heme proteins like P450s1. Intensities of Raman active vibrations can be greatly enhanced
when the interrogating laser beam has a wavelength that
coincides with an electronic transition of the sample. Furthermore the adsorbance of the sample on a metal surface
enhances sensitivity and selectivity to a certain part of the
protein. In this study the heme domain of CYP102 was expressed, purified and substrate interactions were studied
with SERRS. By using a Raman microscope, the sample
volume could be minimised to microliters, opening possi-
S85
residues of P45017α was performed with EDC in 50mM
glycinamide solution at pH 5. The positions of modified
amino acid residues of P45017α were deduced from the
tryptic peptide mappings using MALDI TOF mass-spectrometry (Brucker Bioflex).
More than 20 times repetition of the mass-spectrometric
measurements confirmed that 16 lysine residues were acetylated in detergent solubilized P45017α but some of them
could not be acetylated in the proteoliposomes. Those nonacetylated lysine residues were located near both N and C-terminals. We could identify 10 glycinamidated acidic residues
in the detergent solubilized P45017α but a few of them were
not modified in the proteoliposomes. The nonmodified acidic amino acid residues were located in or near the F-G loop.
The difference in the reactivities of amino acid residues
of P45017α between in the detergent solubilized state and
in the proteoliposomes must be depending on the accessibility of the water-soluble modification reagents to the residues. The nonmodified residues in the proteoliposomes
must be at or near the membrane binding domains.
The present study revealed the existence of the membrane binding domains at N- and C-terminals and F-G loop.
The combination of mass-spectrometry and chemical modification is a powerful and convenient method for the study
of membrane topology of the proteins.
tase (AdR) and of their complexes - were obtained on two
surface types, graphite and mica. The image heights on graphite/mica were 2.2±0.3 nm and 2.6±0.3 nm/2.7±0.3 nm for
the P450scc monomers and aggregates, respectively;
1.8±0.3 nm/1.4±0.3 nm and 2.1±0.3nm/2.2±0.3 nm for Ad
and AdR, respectively. It was shown that AFM enables to reveal and visualize complexes formed between the water-soluble proteins (Ad/AdR) and, also, between the membranebound and membrane-soluble proteins (P450scc/Ad,
P450scc/AdR). The heights of the binary AdR/P450scc,
AdR/Ad and Ad/P450scc were found to be, respectively,
3.2-3.4 nm, 3.1-3.4 nm and 2.8-3.6 nm.
MP21 THE ROLE OF FERRIC-PEROXO
REACTIVITY AT THE
CROSSROADS OF P450
CATALYSIS
THOMAS M. MAKRISa, ILIA G. DENISOVb,
ILME SCHLICHTINGc, and
STEPHEN G. SLIGARa,b
a
The Center for Biophysics, and the bDepartments of Biochemistry, Chemistry, and the College of Medicine, University of Illinois, 505 S. Goodwin Avenue, Urbana,
IL 61801 USA; cDepartment of Molecular Biochemistry,
Max Planck Institute for Molecular Physiology, OttoHahn-Straße 11, D-44227 Dortmund, Germany.
[email protected]
MP20 THE AFM STUDY OF COMPLEX
FORMATION WITHIN THE
CYTOCHROME
P450SCC-CONTAINING SYSTEM
The peroxo-ferric species serves as a critical junction in
not only the P450 monoxygenation scheme, but in the oxidative transformations of a whole host of heme and nonheme metalloenzymes. The robustness of the ferric-peroxo
intermediates, in both protonated and unprotonated forms,
manifests itself in a veritable complexity of fates in the
P450 catalytic cycle. Former studies have implicated the
peroxo-species as (1) an intermediate on the pathway of
Compound I formation (2) a branchpoint for productive
and peroxide evolving chemistries, and (3) a direct oxidant
in a number of electrophilic and nucleophilic oxidations.
Our recent results utilize cryoradiolytic methodologies for
the direct visualization of peroxo-iron evolution as a function of annealing temperature in native and a series of
active site mutant P450s. This has allowed the direct separation of reaction pathways for the discrete heme-oxygen
intermediate involved. Furthermore, in the combinatorial
use of active-site mutants with discriminant reactivity patterns, one can control both the relative stoichiometry of
each of these fates and ascertain the role of each intermediate in various P450 catalyzed oxidation reactions. This
regulation of peroxo reactivity is directly linked to finelytuned „proton wires“, elucidated by both structural and
spectroscopic measurements. Supported in part by National Institutes of Health grants GM31756 and GM33775.
VADIM KUZNETSOVa, YURI IVANOVa,
SERGEY USANOVb, and ALEXANDER
ARCHAKOVa
a
V. N. Orekhovich Institute of Biomedical Chemistry RAMS,
Pogodinskaya Str., 10, Moscow, Russia, bInstitute of Bioorganic Chemistry BAS, Minsk, Belarussia,
[email protected]
An application of atomic force microscopy (AFM) for
the revelation and identification of individual molecules and
their complexes within the monooxygenase cytochrome
P450scc system was demonstrated. The binary complexes
were differentiated from the individual molecules based on
the height parameter. The experiments were carried out
using the direct adsorption method. As supports were used
the hydrophobic surface of highly oriented pyrolytic graphite (HOPG) and the hydrophilic surface of mica. All AFM
experiments were carried out in a tapping mode on a Solver
P47H microscope (NT-MDT).
The AFM images of molecules constituting the P450scc
system - i.e. those monomeric and aggregated cytochrome
P450scc (P450scc), adrenodoxin (Ad), adrenodoxin-reduc-
S86
MP22 CYP2D6: ROLE OF GLU-216 AND
ASP-301 IN QUINIDINE BINDING
MP23 CRYSTALLOGRAPHIC STUDIES
OF PERDEUTERATED P450CAM
MCLAUGHLIN L.A.a, SUTCLIFFE, M.J.b,
PAINE, M.J.I.a, ROBERTS, G.C.K.c, and
WOLF C.R.a,d
FLORA MEILLEURa, MARIE-T DAUVERGNEa,
MICHAEL HAERTLEINb, ILME SCHLICHTINGc, andDEAN MYLESa
a
a
Biomedical Research Centre, University of Dundee, Dundee DD1 9SY, UK, [email protected], bDepartment of Biochemistry and Department of Chemistry, University of Leicester, Leicester LE1 7RH, UK, cBiological
NMR Centre and Department of Biochemistry, University
of Leicester, P.O. Box 138, Leicester LE1 9HN, UK,
d
Cancer Research UK Molecular Pharmacology Unit, Biomedical Research Centre, University of Dundee, Dundee
DD1 9SY, UK.
EMBL, Grenoble-Outstation, BP 181, 38042 Grenoble Cedex 9, France, bILL, 6, Rue Jules Horowitz, BP 156, 38042
Grenoble Cedex 9, France, cMPI, Jahnstrasse 29, 69120
Heidelberg, Germany
[email protected]
Cytochrome P450cam from the soil bacteria Pseudomonas Putida is the monooxygenase responsible for the
stereo- and regiospecific hydroxylation of camphor when
used as sole carbon source. P450cam is the model system
to study structure function relationships in P450s because
of its ready availability, aqueous solubility and relative
ease of purification.
The mechanism of activation of the heme bound dioxygen is still unclear, though a rather complicated proton delivery pathway is likely to be involved. Unfortunately, hydrogen atoms cannot be seen at all in the available X-ray
crystal structures. Neutron crystallography would help determine the key hydrogen atom positions in the protein, but
preliminary experiments show that the large hydrogen incoherent scattering background seriously reduces the signal to noise. This problem can be overcome by deuterating
the sample, either partially, by soaking crystals in D2O, or
more fully, by preparing completely (per)deuterated protein samples.
We have developed protocols for over-expression of
perdeuterated P450cam in high yield using fully deuterated
minimal media and high cell density culture techniques.
Mass spectrometry shows that deuteration of the protein is
greater than 99%. X-ray quality crystals have been grown
and the structure of perdeuterated P450cam determined in
both P43212 and P212121 crystal forms at 1.7 and 2.0 Å resolution, respectively. No significant structural changes
were detected between the hydrogenated and perdeuterated
forms. We have now determined the X-ray structure of
a perdeuterated P450cam-CN- bound complex that mimics
the P450cam-O2 bound intermediary state. The cyanide
complex is structurally identical to the oxy complex, in
particular the two water molecules observed in the
P450cam-O2 complex are there. Neutron diffraction analysis of these crystals should now reveal their protonation
state and the location of the hydrogens.
We have shown recently, directed by our structural
modeling studies, that the CYP2D6 active site residues
Asp-301 and Glu-216 play a key role in the recognition of
basic nitrogen containing substrates. We have further examined the effect of mutations to these residues on interactions with the protypical 2D6 inhibitor, quinidine. Removal
of the negative charge from either or both residues reduced
inhibition of bufuralol 1’ hydroxylation and dextromethorphan O-demethylation by at least 100 fold. Concomitantly, major alterations in binding were obvious from
changes in optical binding spectra. Removing the negative
charge from residue 216 and/or 301 led to a shift in the position of the Amax from 390 nm to ~404 nm and Amin, from
419 nm to ~426 nm. Apparent Kd s of wild type, E216D and
D301E were in the range 0.25-0.50 µM, while single nonconservative substitutions resulted in 30 to 64-fold increases in Kd. The double E216Q/D301Q mutant exhibited the
largest decrease in quinidine binding with an apparent
Kd of 65 µM. Interestingly, the relaxation in binding affinity was associated with the appearance of new metabolites following incubations with quinidine. D301Q and
D301N produced small amounts of 3-hydroxyquinidine,
D301A and D301F mutants produced O-demethylated quinidine, and E216Q/D301Q produced both metabolites.
The implications with respect to CYP2D6 structure and
function are discussed.
S87
MP24 CRYSTAL STRUCTURES OF
EPOTHILONE-D BOUND,
EPOTHILONE-B BOUND, AND
SUBSTRATE-FREE FORMS OF
CYTOCHROME P450EPOK
heme prosthetic group. The crystal structures of epothilone-bound forms reveal that minor but significant structural change has occurred upon epothilone binding. The
epothilones are positioned with its macrolide ring roughly
perpendicular to the heme plane and makes a number of
hydrogen bonds, π−π stacking interaction between thiazole
ring and Tyr96, and hydrophobic interactions. These new
structures provide useful information to understand epothilone-tubulin interaction as well as substrate incorporation
and recognition by P450 enzyme family.
SHINGO NAGANOa, HUIYING LIa,
HIDEAKI SHIMIZUa, CLINTON NISHIDAb, HIROSHI
OGURAb, PAUL R. ORTIZ DE MONTELLANOb and
THOMAS L. POULOSa
REFERENCES
a
Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine California 92697-3900
USA, [email protected], bUniversity of California, San
Francisco, Department of Pharmaceutical Chemistry, 513
Parnassus Avenue, Box 0446, San Francisco CA 941430446 USA
1 Nicolaou K.C., Roschangar F., Vourloumis D. Angew.
Chem. Int. Ed., 37, 2014, (1998).
2 Tang L., Shah S., Chung L., Carney J., Katz L.,
Khosla C., Julien, B., Science, 287, 640, (2000).
Epothilones1 are potential anticancer drugs that stabilize microtubules by binding to tubulin in a manner similar
to taxol. Epothilones are produced by a series of enzymes2
including polyketide synthases (PKSs), a nonribosomal
peptide synthethase (NRPS) and P450epoK heme containing monooxygenase in the myxobacterium Soranigiun
celluosum. P450epoK catalyzes the epoxidation of epothilone C and D, which are synthesized by PKSs and NRPS,
into epothilone A and B, respectively (Fig. 1). The 2.10-,
1.93, and 2.65-Å crystal structures reported here for epothilone D- and B-bound, and substrate-free forms are the
first crystal structures of epothilone binding protein. The
substrate-free P450epoK exhibits triangular P450 fold with
a large vacant substrate-binding pocket just above the
MP25 IDENTIFICATION OF THE
SUBSTRATE-CONTACT
RESIDUES IN CYTOCHROME
P450 27A1
DILYARA MURTAZINAa, SANDRA GRAHAMb,
JULIAN A. PETERSONb, INGEMAR BJORKHEMc,
AND IRINA PIKULEVAa
a
of Texas Medical Branch, 301 University Blvd., Galveston,
TX 77555-1031, USA, bDepartment of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry
Hines Blvd., Dallas, TX 75390-9038, and cDepartment of
Clinical Chemistry, Karolinska Institute, Huddinge Hospital, S-141 88 Huddinge, Sweden
Cytochrome P450 27A1 (P450 27A1) plays an important role in cholesterol degradation to bile acids. This enzyme catalyzes multiple oxidation reactions at the C-27
atom of steroids in the classical (hepatic) bile acid biosynthetic pathway and cholesterol in the alternative (peripheral) pathway. Little is known about the mode of interaction
of P450 27A1 with different substrates. The present study
utilized heterologous expression in E. coli and site-directed
mutagenesis to identify substrate-contact residues in P450
27A1. Targets for mutation were selected based on a substrate-bound computer model of P450 27A1 that was constructed based on the crystal structure of P450 BM-P. We
also capitalized on the observation that determinants of
substrate specificity in P450s whose structures have been
solved by X-ray crystallography are located at similar
alignment positions to hold substrate in place within the
active site. Twenty alignment positions that encompass
four secondary structural elements that form the sides of
the active site in structurally characterized P450 were investigated. Generally two substitutions, one to a smaller-
Fig. 1.
S88
-sized residue and the other one is to a lager-sized residue,
were introduced at each alignment position under study.
Totally more than 30 mutants were generated. Interaction
of P450 27A1 wild type and mutants with one of the enzyme’s substrates 5β-cholestane-3α, 7α, 12α-triol was evaluated by using spectral binding assay. The apparent binding constants of the H103F, T110V, M301C, V304L,
V367L, N370L, V482A, L283I, L483I, and L483V mutants were increased 16-80-fold indicating that the mutated
residues are likely involved in the interaction with this substrate. Investigation of the catalytic properties and product
profiles of the mutants that showed significantly impaired
binding of the substrate is in progress.
MP27 ALTERATION OF CYP 4A4
REGIOSPECIFICITY BY
SITE-DIRECTED MUTAGENESIS
OF RESIDUES IN THE
SUBSTRATE BINDING
CHANNEL
LINDA J. ROMAN, MELISSA DE LA GARZA,
MARIBETH MOCK, DAWN HARRIS, and
BETTIE SUE MASTERS
The University of Texas Health Science Center, San Antonio, TX 78229; [email protected]
MP26 METABOLISM PREDICTION
FOR CYTOCHROME P450
In contrast to all other P450s which hydroxylate eicosanoids and/or fatty acids at the ω-1, ω-2, or ω-3 positions1,2, P4504As very specifically catalyze hydroxylation at
the more energetically unfavorable omega carbon3,4. It is
possible that steric restriction of the substrate access channel near the heme of the CYP 4As is responsible for limiting hydroxylation to the omega carbon. Modeling studies
of CYP4A115,6 identify several residues deep in the pocket
that may be responsible for this characteristic. Residue
E321 of CYP 4A4 appears to be positioned near the bottom
of the channel and thus controls its depth. We mutated
E321 to alanine and found that in PGE1 metabolism, the
ratio of ω:(ω-1) products was decreased, i.e., more (ω-1)
PGE1 was formed. Another interesting residue at the bottom of the substrate-binding channel of CYP 4A4 is L132;
this residue has been mutated to phenylalanine (as in
CYP102, which makes only (ω-1,2 or 3) products) and the
resultant protein is being characterized. Additional residues potentially involved in substrate binding or in defining the shape of this channel are being mutated for future
studies.
ISMAEL ZAMORAa, MARIANNE RIDDERSTRÖMb,
RICCARDO VIANELLOc, GABRIELE CRUCIANId,
TOMMY B. and ERSSONb
a
Lead Molecular Design, s.l. ,Vallés 96-102(Loc27), 08190
Sant Cugat del Vallés, Spain, b DMPK & BAC, AstraZeneca
R&D Mölndal, S-431 83 Möndal, Sweden, c Molecular Dsicovery, Ltd., 4 Chandos Street W1A 3BQ London, UK, d Perugia University, Via Elce Di Soto 10, I-06123 Perugia,
Italy
The use of computational methods to predict the site of
metabolism and the enzyme responsible of the reaction
enables the possibility to optimize the drug metabolism
properties of virtual compounds in the drug discovery process, reducing the lead optimization cycle time. The aim
of this work is to present a method able to predict the site
of metabolism based on a pharmacophoric representation
of the GRID molecular interaction fields computed on the
protein models, a pharmacophore fingerprint calculated on
the substrates and considering the chemical reactivity. The
technique is not limited to GRID molecular interaction
fields, but any type of fields can be used. In this sense a field representing the selective interaction between the different cytochromes was used to describe the protein responsible for the metabolic reaction. The selective interactions
were obtained from an analysis on CYP1A2, 2C9, 2C19,
2D6, and 3A4. The methodology was used on a set of
90 reactions catalyzed by CYP2C9, 50 for CYP2D6 and
180 for the CYP3A4 substrates. The rate of good prediction for the site of metabolism was over 75 % for all the
cytochromes.
REFERENCES
1. Ho P.P., Fulco A.J.: Biochim. Biophys Acta, 431, 249
(1976).
2. Fukuda T., Imai Y., Komori M., Nakamura M., Kusunose E., Satouchi K., Kusunose M.: J. Biochem (Tokyo), 115, 338 (1994).
3. Johnson E.F., Walker D.L., Griffin K.J., Clark J.E.,
Okita R.T., Muerhoff A.S., Masters B.S.: Biochemistry,
29, 873 (1990).
4. Roman L.J., Palmer C.N.A., Clark J.W., Muerhoff A.S.,
Griffin K.J., Johnson E.F., Masters, B.S.S.:Arch. Biochem. Biophys., 307, 57 (1993) .
5. Lewis D.F.V., Lake, B.G.: Xenobiotica 29, 763 (1999).
6. Chang Y.-T., Loew G.H.: Proteins: Structure, Function,
and Genetics 34, 403 (1999).
S89
MP28 CRYSTAL STRUCTURE OF
PUTIDAREDOXIN
MP29 THIOLS PREVENT P420
FORMATION AND
AGGREGATION IN
25-HYDROXYVITAMIN D3
24-HYDROXYLASE
IRINA SEVRIOUKOVA, CARLOS GARCIA, HUIYING
LI, B. BHASKAR, and THOMAS POULOS
Department of Molecular Biology and Biochemistry,
University of California Irvine, Irvine CA 92697, USA,
[email protected]
MICHAEL J. THEISENa, ANDREW ANNALORAb,
SABRINA A. BERETTAa, ANDRZEJ PASTUSZYNb,
EDMUND D. MATAYOSHIa, JOHN L. OMDAHLb, and
MARK L. CHIUa
Putidaredoxin (Pdx) from Pseudomonas putida shuttles
as one-electron donor/acceptor between NADH-dependent
FAD-containing putidaredoxin reductase and the terminal
oxygenase cytochrome P450cam (P450cam, CYP101).
Despite many efforts, Pdx has resisted crystallization. Pochapsky and co-workers have applied NMR methodology
to obtain a partial structure of Pdx where the [2Fe-2S] cluster, which could not be defined due to the paramagnetic
broadening effect, was modeled using the crystal structure
of Anabena ferredoxin1,2. Later, the Pdx model was refined
using a gallium-substituted apoprotein3 and modeling based on the crystal structure of bovine Adx 4. In addition,
multidimensional NMR methods and NMR relaxation measurements were utilized to investigate structural changes
that occur upon reduction of the metal center in Pdx5,6.
This hybrid structure of Pdx has served as a guide in the
structure/function studies on Pdx for many research
groups. However, the accuracy of the model has to be evaluated and this could be achieved by determining the crystal structure of the protein. For this reason, we have prepared Cys73Ser and Cys73Ser/Cys85Ser mutants to improve Pdx stability and crystallized and solved the structures of the mutant proteins to 1.65 and 1.47Å, respectively.
Our structural data revealed the precise environment of the
[2Fe-2S] cluster in Pdx that differs from that modeled in
the NMR structure. Dissimilarity between the NMR and
x-ray structures is also observed in the (αhelical and C-terminal domains of the protein. We will discuss these structural differences and their implications for the Pdx-redox
partner interactions and electron transfer.
a
Department of Structural Biology, Abbott Laboratories,
Abbott Park, Illinois, USA, bDepartment of Biochemistry
and Molecular Biology, University of New Mexico School
of Medicine, Albuquerque, New Mexico, USA
[email protected]
Calcium homeostasis is affected by levels of the hormone 1,25-hydroxyvitamin D3 (1,25OH-D3).1 The membrane-associated, mitochondrial cytochrome P450C24
(C24) deactivates 1,25OH-D3 by hydroxylation at the 24position, and the inactive hormone is subsequently further
modified and excreted. Understanding C24 activity is important for therapeutic efforts to modulate calcium levels.
In vitro experiments reveal that recombinant rat C24 forms
large aggregates concomitant with the P450 to P420 transition. Both phenomena are prevented by the presence of thiols. Interestingly, thiols do not perturb the C24 spectrum,
suggesting that they do not bind in the active site, unlike
what is observed with P450cam.2 Various biophysical characterizations demonstrate no major differences between
the P450 and P420 states, nor any effect of thiols on the
protein tertiary structure. It is suggested that one or more
surface cysteines may cause nonspecific aggregation that
predisposes C24 to convert to the P420 form. With
P450cam, such a reactive surface cysteine was found to
cause a mixed population of dimers and monomers, however there was no suggestion of larger aggregates or correlation with P420 formation.3
REFERENCES
REFERENCES
1. Pochapsky T. C., Ye X. M: Biochemistry 30, 3850
(1991).
2. Pochapsky T. C., Ye X. M., Ratnaswamy G., Lyons T. A.:
Biochemistry 33, 6424 (1994).
3. Pochapsky T. C., Kuti M., Kazanis S.: J. Biomol. NMR
12, 407 (1998).
4. Pochapsky T. C., Jain N. U., Kuti M., Lyons T. A., Heymont J.: Biochemistry 38, 4681 (1999).
5. Lyons T. A., Ratnaswamy G., Pochapsky T. C.: Prot.
Sci. 5, 627 (1996).
6. Sari N., Holden M. J., Mayhew M. P., Vilker V. L.,
Coxon B. Biochemistry 38, 9862 (1999).
1. Omdahl J.L., Bobrovnikova E.V, Annalora A., Chen P.,
Serda R.: J. Cell. Biochem. 88, 356 (2003).
2. Sono M., Andersson L.A., Dawson J.H.: J. Biol. Chem.
257, 8308 (1982).
3. Nickerson D.P., Wong, L.L.: Protein Eng. 10, 1357
(1997).
S90
MP30 THE SELECTIVE INHIBITION
OF CYTOCHROME P450 1
ENZYMES BY THE
DERIVATIVES OF
RUTAECARPINE
MP31 THE PROTEIN STABILIZATION
OF COUMARIN
7-HYDROXYLASE BY ITS OWN
INHIBITORS
PIRKKO VIITALAa, OLAVI PELKONENa, and
RISTO JUVONENb
YUNE-FANG UENGa, MING-JAW DONa, DAVID
F. V. LEWISb, SHU-YUN WANGa, and MEI-WEN, TSAIa
a
of Oulu, Oulu, Finland, bDepartment of Pharmacology and
Toxicology, University of Kuopio, Kuopio, Finland
[email protected]
a
National Research Institute of Chinese Medicine, 155-1
Li-Nong Street, Sec. 2, Taipei 112, Taiwan, R. O. C. and
b
School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, UK
The murine CYP2A5 expression is regulated in a unique fashion compared with other CYP forms. It is inducible by phenobarbital (PB), but also several structurally
unrelated chemicals induce it. As far as the mechanisms of
CYP2A5 induction are concerned, receptor-mediated induction of CYP2A5 is essentially ruled out due to the wide
variation in the structures of the chemicals that act as COH
(coumarin 7-hydroxylase) inducers. In most induction studies the increases of CYP2A5 correlate well with the elevation of the corresponding mRNA levels which may be
due to an increased transcription of Cyp2a5 gene or stabilization of mRNA.
Some inhibitors of P450 enzymes can elevate its enzyme activity by stabilizing the protein. In this work we
have studied effects of potent COH inhibitors such as γ-lactones, tranylcypromine, metyrapone and pilocarpine on
COH enzyme activity in mouse primary hepatocytes. Lactone derivatives coumarin, γ-nonanoic lactone, γ-phenyl- γbutyrolactone, 7-methylcoumarin and 7-amino-4-methylcoumarin did not cause any induction of COH, PROD or
EROD activity. In contrast to lactones other COH inhibitors such as tranylcypromine, metyrapone and pilocarpine
increased COH activity about 5-fold, but had no effect on
CYP2A5 mRNA levels. Metyrapone induced PROD activity 3.5-fold. N-atom containing inhibitors induced COH
activity but did not increase mRNA level suggesting that
they could stabilize CYP2A5 protein. Nitrogen seems to
act a significant role, because other COH inhibitors were
not capable of affecting COH activity. Apparent induction
of CYP2A5-associated COH activity on the basis of protein stabilization represents another mechanism of regulation of this enzyme.
Three cytochrome P450 (CYP) members, CYP1A1,
CYP1A2, and CYP1B1, have been identified in human liver microsomes and show importance in the activation and
detoxication of a number of endogenous and exogenous
compounds. Our previous report showed that the alkaloid
rutaecarpine selectively inhibited CYP1A2 in mouse and
human liver microsomes. To have potent and selective inhibition of human CYP1 members, methoxyl and halogenated derivatives of rutaecarpine were synthesized. Structural modelling shows a good fitting of rutaecarpine with
the putative active site of human CYP1A2. Two hydrogen
bonds can be formed between the keto- and N14-groups of
rutaecarpine and the Thr208 and Thr473 residues of CYP1A2,
respectively. The C-ring moiety of rutaecarpine forms π-π
stacking interaction with the aromatic ring of Phe 205 residue
of CYP1A2. 7-Ethoxyresorufin O-deethylation activities
of Escherichia coli membranes expressing bicistronic human CYP1 members were determined. 7,8-Dehydrorutaecarpine with C7-C8 double bond in the C-ring strongly inhibited CYP1A1, CYP1A2, and CYP1B1 catalytic activities but without selectivity. Among the derivatives, 10- and
11-methoxyrutaecarpine are the most selective CYP1B1
inhibitors. 1-Methoxyrutaecarpine and 1,2-dimethoxyrutaecarpine are the most selective CYP1A2 inhibitors. However, the selectivity was not improved by the introduction of
halogen groups. (Supported by National Research Institute
of Chinese Medicine and NSC91-2320-B-077-010)
REFERENCES
1. Ueng Y. F., Jan W. C., Lin L. C., Chen T. L., Guengerich F. P., Chen, C. F., Drug Metab. Dispos. 30, 349,
(2002).
REFERENCES
1. Viitala P., Posti K., Lindfors A., Pelkonen O., Raunio
H.: Biochem. Biophys. Res. Commun. 280, 761 (2001).
S91
structure choice. Conserved and variable zones in the
structures of CYP templates can be identified. CSBs were
then aligned against the CYP3A4 target sequence using inhouse tools designed for multiple profile alignment, to generate about 58,000 distance constraints and 760 dihedral
constraints for the whole structure rebuilding. This set of
CSB and the corresponding constraints dataset can be used
as a robust framework to rebuild the isoforms of the whole
CYP 3A family in human and mammals.
MP32 VALIDATION OF A 3D-REBUILT
P450 3A4 STRUCTURE WITH
CYCLOPEPTIDE METABOLISM :
IMPLEMENTATION OF NEW
TECHNIQUES INVOLVING
SOFT-RESTRAINED DYNAMICS
DOCKING SIMULATIONS GIVES
NEW INSIGHTS ON THE
MULTIPLE SUBSTRATE
SPECIFICITY
Information drawn from the 3D rebuilt structure:
The CYP3A4 structural model was submitted to various molecular dynamics (MD) simulations, either for checking the stability of the structure without the building distance constraints, or to verify the consistency of the model with the various mutants of the literature. Biochemical
data of substrate interaction and metabolism obtained at
the laboratory were interpreted by submitting the 3D model to MD simulations in the presence of various substrates
such as testosterone or cyclotetrapeptides. Simulations
were performed with the substrate positioned initially out
of the protein structure, and not within the heminic active
site. with no initial guess in the orientation of the ligand, as
it is free to evolve during the MD simulations. Soft distance constraints were applied to the substrate to guide it
towards the heme, so that different entrances and pathways
can be examined through the simulations. All the simulation results showed that:
1) two different gates in the enzyme structure proved to
be accessible, depending on the substrate, with respect to
its size or chemical structure,
2) several interacting helices play a crucial role in the
gating and the protection of the active site after substrate
entrance,
3) at least two substrate molecules can be channeled towards the active site(testosterone/α- naphtoflavone cooperativity has been studied in details),
4) energetic profiles can be estimated from the force field parameters and account for variations in the access to
the active site, 5) the final positions of the ligand in the vicinity of the heme (distance Fe-substrate) were found in
agreement with experimental metabolism data of the natural cyclotetrapeptide phytotoxin tentoxin and some of its
natural derivatives2.
Thus, the rebuilt structure seemed to account for the
multi-specific features of CYP3A4 isoform, in terms of
substrate channeling, and allosteric behavior. This is notable, as many drugs in the past have been withdrawn from
the market or stopped in clinical trials due to adverse side
effects from their interactions with these enzymes. This is,
to our knowledge, the first model of CYP3A4 available
with such predictive capability3.
FRANÇOIS ANDRÉa, NICOLAS LOISEAUb, and
MARCEL DELAFORGEa
a
CNRS URA 2096, Département de Biologie Joliot-Curie,
CEA-Saclay, 91191 Gif-sur-Yvette cedex, France, bINRA
UR66, Laboratoire Pharmacologie Toxicologie, 31931
Toulouse Cedex 9, France, [email protected]
CYP3A4 is the most important family member of drug metabolizing enzymes in humans, as it affects as many as 50% of
all known drugs interacting with this form of cytochrome
P450. However, it is still one the most poorly understood
member within the CYP family, with respect to its drug metabolizing action. The absence of any crystal structure available
in the PDB or in the literature represents a major problem in
drug development, which can be overcome by rebuilding methods such as comparative modeling techniques, based on robust distance geometry protocols for structure optimization.
We propose, in this study, to validate the CYP3A4 isoform
3D model rebuilt at the laboratory using in-house modeling
3D alignment tools1, by analyzing the drug pathway through
different possible clefts in the structure and towards the heme
active site. For this purpose, we implemented techniques
issued from molecular dynamics simulations with a special
attention to drag the substrate from the outer medium under
soft-restrained conditions. This new approach allows to take
into account the flexibility of the protein, a prevalent feature
in the CYP3A4 structure, as well as the flexibility of the
ligand, and the relationship between its size and chemical
structure and the potential entrance site in the enzyme.
Rebuilding method:
To rebuild the structure of CYP3A4, six high-resolution X-ray structures of bacterial, fungal and mammalian
CYPs were selected to form the initial multiple template.
These structures displayed low identity (about 20 % in average), with significant parts in the N-terminal region that
are not conserved and cannot be aligned by standard multiple primary sequence alignment methods. The interest of
our method resides in the use of a local structural alignment tool (GOK ABI, Paris) in the internal coordinates
space, based on phi/psi or alpha/tau analysis, that allows to
determine common structural blocks (CSB) with no pivot
REFERENCES
1. Minoletti M., Santolini J., Haraux F., Pothier J., André
F.: Proteins 49, 302 (2002)
S92
the ω orientation of the substrates with respect to the heme
iron atom was clearly preferential in the MD simulations.
On the other hand, the activation barrier calculated for hydrogen atom abstraction on the ω position by a neutral hydroxyl radical5 was higher than that for the other positions,
which would shift the overall product formation away from
ω hydroxylation. The calculated barrier heights are very
sensitive to the electrostatic environment.
In summary, it is concluded that the combination of simulation and analysis of the dynamics, and quantum mechanical calculations is a valuable tool to investigate and
understand substrate binding and product formation.
2. Delaforge M., André F., et al.: Eur. J. Biochem. 250,
150 (1997).
3. Provisional US patent application n° US 60/421,569
US. A method for performing restrained dynamics
docking of one or multiple substrates on multi-specific
enzymes
MP33 SUBSTRATE DYNAMICS IN
CYTOCHROME P450-BM3
- DYNAMICS OF SUBSTRATE
BINDING BY AUTOMATED
DOCKING AND MOLECULAR
DYNAMICS SIMULATION
REFERENCES
1. Jones G., Willett P., Glen R.C., Leach A.R., Taylor R.:
J. Mol. Biol. 267, 727 (1997).
2. Lindahl E., Hess B., Van der Spoel D.: J. Mol. Mod. 7
306, (2001).
3. Van Gunsteren W.F., Billeter S.R., Eising A.A., Hünenberger P.H., Krüger P., Mark A.E., Scott W.R.P., Tironi
I.G.: Biomolecular Simulation: The GROMOS96 Manual and User Guide, Vdf Hochschulverlag AG ETH
Zürich, Zürich, Switzerland (1996).
4. Amadei A., Linssen A.B.M., Berendsen H.J.C.: Proteins: Struct. Funct. Gen. 17, 412 (1993).
5. Jones J.P., Mysinger M., Korzekwa K.R.: Drug Metab.
Disp. 30, 7 (2002).
K. ANTON FEENSTRA, CHRIS DE GRAAF, JEWGENI
STARIKOW, JAN N.M. COMMANDEUR, and
NICO P.E. VERMEULEN
Vrije Universiteit Amsterdam, Sect. Molecular Toxicology,
Dept. Pharmacochemistry, [email protected],
www.few.vu.nl/~feenstra
Cytochrome P450 BM3 (Cyp102) from Bacillus megaterium catalyses the hydroxylation of fatty acids. Several
mutants have been described that also hydroxylate alkanes,
like octane. Hydroxylation takes place on sub-terminal positions (ω-1, ω-2, ω-3), but not on the terminal (ω) position. The interactions between enzyme and substrate are
very subtle; there are few specific ionic or hydrogen bonding interactions, and no ‘complementary fit’ of the substrate in the binding cavity. Using a combination of computational methods, these interactions and differences between mutants are investigated. Binding modes are found
from automated docking, additional dynamics and binding
statistics come from Molecular Dynamics (MD) simulations. Additionally, as a step towards accurate prediction of
product ratio’s, intrinsic reactivities are obtained from
Quantum Mechanical (QM) calculations of the substrate
transition state complex.
Substrates were docked into the proteins using the Gold
automated docking program1. MD simulations were started
from several most dissimilar binding orientations, each for
a period of 10ns, using the Gromacs MD package 2 and the
Gromos forcefield3. Wild-type and mutant dynamical features are characterized using Essential Dynamics (ED) analysis4.
Qualitative differences in dynamical behaviour, as seen
in the ED analysis, between wild-type and some of the mutants were observed, both for the dynamics of the whole
enzyme and for the active site region only. Binding of the
substrates was found to be highly dynamical, the substrates
have much freedom to move around the active site interior
and bind transiently at different positions, sometimes for
relatively long times of several nanoseconds. Statistically,
MP34 A QM/MM STUDY OF
CYTOCHROME P450:
THE INFLUENCE OF THE
ENZYME ENVIRONMENT ON
SPECIFIC STEPS IN THE
CATALYTIC CYCLE
A.R. GROENHOF, M. SWART, A.W. EHLERS, and
K. LAMMERTSMA
Department of Organic and Inorganic Chemistry, Division
of Chemistry, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands, [email protected]
Cytochromes P450 catalyze the stereospecific addition
of molecular oxygen to nonactivated hydrocarbons and
therefore perform many vital bioregulatory functions. The
mechanism of this monooxygenation reaction has been studied extensively, but remains a subject of discussion and
debate. Also many quantum chemical studies have been
devoted to this topic, but few take the enzyme environment
explicitly into account.
In this contribution, we report a QM/MM study on the
active site of P450cam in which its protein environment is
S93
for the Mycabacterium sp. In both cases proteins with
CYP51 activity have been isolated. This led to a conclusion
that CYP51 has developed very early in the cytochrome
P450 evolution and was transfered vertically from prokaryotes to eukaryotes. A putative CYP51 was found also in
Streptomyces coelicor, but the sterol synthesis in this prokaryote has not yet been clearly demonstrated. Construction
of a phylogenetic tree containing CYP51 genes from all so
far available eukaryotic and prokaryotic sequences gave the
topology ((bacteria,plant)(animals,fungi)), which differs
from the previousy proposed topology (bacteria(plant(animals,fungi)))1. In order to evaluate the credibility of the
proposed novel CYP51 filogeny, all available bacterial cytochrome P450 sequences have been analyzed. Mycobacterium and Methylococcus capsulatus CYP51s clustered together in the bacterial CYP dendrogram, however, the
Streptomyces coelicor CYP51 locates in a distant branch,
suggesting that S. coelicor might not originate from
a CYP51 ancestor. The ((bacteria,plant)(animal,fungi)) topology of the CYP51 cluster has been preserved after the
closest bacterial neighbours to the Mycobacterium/Methylococcus cluster have been added. This indicates again that
the Mycobacterium/Methylococcus CYP51s are closer to
plant CYP51s as compared to any other bacterial cytochrome P450. Available biochemical data support a close
relation between bacterial and plant CYP51s. The Mycobacterium tuberculosis CYP51 enzyme is similar to plant
CYP51, using obtusifoliol as a preferred substrate2. These
data lead us to propose that Mycobaterium sp. and Methylococcus capsulatus CYP51 genes were gained through
a lateral gene transfer from plants. We propose further that
Streptomyces coelicor CYP51 belongs to another bacterial
CYP group and has at present an incorrect gene family annotation. Additional prove of our hypothesis comes from
a recent independent investigation where the Mycobacterium tuberculosis proteins have been compared against all
bacterial and eukaryotic proteins. This phylogenetic analysis proposed that eight Mycobacterial genes, one of them
being CYP51, have been aquired by a lateral gene transfer
from eukaryotes3.
taken into account. The entire system is split into a QM
part for the active site and a MM part for the enzyme environment. All interactions between the QM and MM parts
are taken into account by using a protein-specific force field (AMBER95) that is also used for the interactions within the MM part. The QM part is described by Density
Functional Theory, which enables the treatment of larger
QM systems, in this case up to approximately 150 atoms.
A recently developed link model (AddRemove) is used for
treating the interactions at the QM/MM boundary.
Results will be presented for specific steps along the
catalytic cycle, with emphasis on the spin states and properties that relate to experimental data. The QM/MM results will be compared with the results for the isolated active site, i.e. iron porphyrin with or without the axial iron
ligands. This enables a separation of the intrinsic properties of the iron porphyrin ring from the properties induced
by the presence of either the axial ligands or the full enzyme environment. Mainly the first steps of the catalytic
cycle will be addressed. For these steps many experimental
data are available to validate the computational setup. The
predictive power of this approach will be explored for the
subsequent steps of the catalytic cycle for which far less
experimental data are available.
MP35 ASPECTS ON EVOLUTION OF
THE CYP51 FAMILY
TADEJA REÎENa, NATA·A DEBELJAKa,
DU·AN KORDI·b, and DAMJANA ROZMANa
a
Institute of Biochemistry, Medical Center for Molecular
Biology, Medical Faculty, University of Ljubljana, Vrazov
trg 2, SI-1000 Ljubljana, Slovenia; bDepartment of Biochemistry and Molecular Biology, JoÏef Stefan Institute, Jamova 39, SI-1000 Ljubljana. [email protected]
Cholesterol is an essential component of eukaryotic
membranes. Its metabolites (steroid hormones, bile acids,
vitamin D and oxysterols) have many different phyisiological functions. A member of the cytochrome P450 superfamily CYP51 (lanosterol 14α-demethylase) participates in
the postsqualene portion of cholesterol biosynthesis and catalyses the removal of the 14α-methyl group of sterols. Additional role of CYP51 is belived to be in overproduction of
MAS (meiosis activating sterol), which are supposed to be
signalling molecules with a role in mammalian reproduction. Enzymes with CYP51 activity are found in three eukaryotic phyla and also in some prokaryotes. The eukaryotic CYP51s use different substrates: lanosterol (mammals
and fungi), 24-methylene-24,25-dihydrolanosterol (fungi)
and obtusifoliol (plants). The presence of sterols in prokaryotes is limited to only some taxonomical groups. Up-todate sterols were found and their synthesis shown for the
Methylococcaceae family (Methylococcus capsulatus) and
REFERENCES
1. Debeljak N. Fink M. Rozman D.: Arch Biochem Biophys. 409,159 (2003)
2. Bellamine A., Mangla A.T., Dennis A.L., Nes W.D.,
Waterman M.R.: J Lipid Res. 42,128 (2001)
3. Gamieldien J., Ptitsyn A., Hide W.: Trends Genet. 18, 5
(2002)
S94
exons are localised in head-to-head orientation in closeness
to the genes of CYP4A11 and CYP4B1. Functional analyses on CYP4Z1 recombinantly expressed in E.coli and generation of monoclonal antibodies are ongoing studies.
MP36 IDENTIFICATION OF NOVEL
HUMAN CYTOCHROME P450S,
CYP4Z1 AND THE
TRANSCRIBED PSEUDOGENE
CYP4Z2P
REFERENCES:
1. Nelson D.R.: The cytochrome P450 homepage.
http://drnelson.ut-mem.edu/CytochromeP450.html
2. Hoch U., Ortiz de Montellano P.R.: J. Biol. Chem. 276,
11339 (2001)
3. Bylund J. et al.: Biochem. Biophys. Res. Commun. 296,
677 (2002)
MICHAEL RIEGERa, REINHARD EBNERb,
ANDREA KIESSLINGa, JACQUES ROHAYEMc,
MARC SCHMITZa, ACHIM TEMMEa,
ERNST PETER RIEBERa, and BERND WEIGLEa
a
Institute of Immunology, Medical Faculty Carl Gustav Carus, Technical University Dresden, Fetscherstrasse 74,
01307 Dresden, Germany, bAvalon Pharmaceuticals, Gaithersburg, Maryland, USA, cInstitute of Virology, Medical
Faculty Carl Gustav Carus, Technical University Dresden,
Germany, [email protected]
MP37 CHARACTERIZATION OF
ARABIDOPSIS THALIANA
CYP711A1
MIYUKI SHIMOJI a, FRANCIS DURSTb,
IRÈNE BENVENISTEb, and RALF MORGENSTERNa
We identified the full length cDNA of a novel human
cytochrome P450 (CYP), CYP4Z1, and of a closely related
pseudogene CYP4Z2P from the breast carcinoma line SKBR-3. They represent members of subfamily 4Z, that have
already been predicted by the CYP450 Nomenclature
Committee1 based on genomic database research. Apart
from expressed sequence tags (ESTs) and partial genomic
sequences, experimental data revealing full length mRNA
sequences were missing so far.
The cDNA of CYP4Z1 (1910 bp) contains an open reading frame of 1515 bp, that codes for a 505 aa protein.
CYP4Z1 meets the characteristics of a functional CYP enzyme with its conserved heme-binding domain, substrate
recognition sites and subcellular localisation signal. The
sequence identity is higher to members of subfamilies
4A (51%) and 4B (45%) than to 4F (40%), but the highest
identity (54%) was found to CYP4X1, a recently identified CYP2. Most of CYP4A, 4B and 4F family members
have a conserved glutamic acid in their I-helix region, that
enables them to bind heme covalently and represents
a unique feature in the CYP 4 family3. In contrast,
CYP4Z1 as well as CYP4X1 contain alanine instead of
glutamic acid at this position. Therefore, 4Z1 and 4X1
might have an exceptional position within family 4. Functional data are required to elucidate the substrate spectrum
of both enzymes.
In contrast to CYP4Z1, a single nucleotide change in
exon 8 of the CYP4Z2P gene causes an early stop codon
that leads to a truncated CYP4Z2 protein of 340 aa lacking
the heme-binding site and the endoplasmic reticulum retention signal KKVC. Thus, it represents a transcribed
CYP pseudogene, consequently named CYP4Z2P. The homology to CYP4Z1 in the first 340 aa is 96%.
The mapping of both new CYP4Z genes on chromosome 1p33 by bioinformatics elucidated the genomic organisation with highly conserved intron/exon boundaries.
The genes for CYP4Z1 and CYP4Z2P consisting of 12
a
Institute of Environmental Medicine, Division of Biochemical Toxicology, Karolinska Institutet, Box 210, S-17177,
Stockholm, Sweden, bIBMP-CNRS UPR2357 Dept. d’ Enzymologie Cellulaire et Moleculaire, 28 rue Goethe
F-67083 Strasbourg, France, [email protected]
We cloned recently several fatty acid ω-hydroxylases
from various plant species which formed the first members
of the CYP86 and CYP94 families1, 2. Phylogenetic analysis
of these P450s showed that they all clustered in one branch
of the „non-A“3 group of plant P450 and that they appeared
closer to animal or fungal fatty acid hydroxylases than to
other plant P450. One of these P450, CYP711A1 which is
located at the base of this „fatty acid hydroxylase branch“
is phylogenetically related to the CYP74 family which uses
fatty acid hydroperoxides as substrates, and to mammalian
CYP5, the thromboxane A synthase. These P450 do not require an electron transfer system for catalytic activity. We
have now cloned CYP711A1 from mRNA of Arabidopsis
thaliana seedlings induced with 1mM methyl jasmonate for
24h. The protein was expressed in WAT11 yeast strain carrying an A. thaliana NADPH-P450 reductase and another
yeast strain expressing the original yeast reductases. The
CO-difference spectra as evidence of functional P450 and
the substrate specificity of CYP711A1 will be presented.
This research was supported in part by Human Frontier Science Program Organization research grant RG0280/1999-M
and Swedish Cancer Society.
REFERENCES
1. Benveniste I., Tijet N., Adas F., Philipps G., Salaün J.P.,
Durst F.: Biochem. Biophys. Res. Commun. 243, 688,
(1998)
S95
dingly decreased p-NPH and AlAT activities to normal levels.
Our experience suggests that the combined administration of caffeine and acetaminophen potentiates acetaminophen hepatotoxicity that should be taken into consideration in developing and using such preparations. As long as
analgesic mixtures containing paracetamol and caffeine are
available, analgesic hepatotoxicity will continue to be
a problem also.
2. Tijet N., Helvig C., Pinot F., Bouquin R., Lesot A.,
Durst F., Salaün J.P., Benveniste I.: Biochem. J. 332,
583, (1998)
3. Shimoji M, Benveiste I, Pinot F, Salaün JP, Wang
Y,Durst F: Biochemistry, Biophysics and Molecular
Biology, 2001, La Grande Motte, France.
MP38 INDUCIBLE CYTOCHROME
P 450 (CYP2E1) BY COMBINED
ADMINISTRATION OF
ACETAMINOPHEN AND
CAFFEINE
MP 39 MOLECULAR MODELING AND
SITE-DIRECTED MUTAGENESES
DEFINING CATALYTIC SITES IN
INSECT P450S METABOLIZING
ALLELOCHEMICALS AND
INSECTICIDES
VALENTINA KOVALENKO, and ALLA VORONINA
Institute of Pharmacology & Toxicology, Academy of Medical Sciences, vul Ezhena Pot’e 14, 03057 Kyiv, Ukraine,
[email protected]
JEROME BAUDRYa, XIANCHUN LIb, ZHIMOU WENc,
LIPING PANc, MAY R. BERENBAUMb, and
MARY A. SCHULERc
CYP consist of a superfamily that play an important
role in the metabolism of a wide variety of xenobiotics,
including drugs, carcinogens and environmental agents, as
well as endogenous compounds such as steroids and fatty
acids. The individuals izoforms have different regulatory
and functional characteristics includingselective substrate
specificities. CYP2E1 is of particular interest because it is
involved in the metabolism activation of many drugs associated with toxic effects. Mixed analgesic preparations
comprising both acetaminophen and caffeine are widely
used. Due to ability of the said substances to induce cytochrome P-450 2E1, the effect of combined administration
of both acetaminophen and caffeine on p-nitrophenol hydroxylase (p-NPH) activity in liver microsomes (a marker of
cytochrome P-450 activity) as well as alanine aminotransferase (AlAT) activity in blood serum was studied using
unbred male mice.
It was shown that acetaminophen administered intragastrically to mice during 3 days once daily at a range of 100350 mg/kg dose dependently elevated the activities of
p-NPH in liver microsomes and AlAT in blood serum. Caffeine administered intragastrically during 3 days once daily at a range of 18-70 mg/kg didn’t affect the activities of
AlAT but p-NPH in liver microsomes dose dependently
increased in fold. However, the combined administration of
both caffeine and acetaminophen significantly increased
the activites of p-NPH in liver microsomes in 4,3 fold compared of normal. An increase in AlAT of 4 fold the upper
limit of normal (ULN) was recorded of pretreatment of
both drugs. p-NPH and AlAT activities were more increased with combined administration of both caffeine and acetaminophen as compared to acetaminophen alone. The high
correlation between both p-NPH and AlAT activities was
observed. Co-administrered composition „MV“ with acetaminophen and caffeine in doses 350 and 70 mg/kg accor-
a
School of Chemical Sciences, bDepartment of Entomology,
and cDepartment of Cell & Structural Biology, University
of Illinois, Urbana, IL, 61801, USA
P450-mediated detoxification strategies are encountered in insects in the evolution of resistances to toxic plant
allelochemicals and synthetic insecticides. Examples of
these include the Papilio polyxenes CYP6B1 protein that
metabolizes toxic furanocoumarins present in its hostplants, the Helicoverpa zea CYP6B8 protein that metabolizes furanocoumarins in its hostplants and insecticides, the
Musca domestica CYP6D1 protein that metabolizes pyrethroid insecticides and polycyclic aromatic hydrobarbons
and several Anopheles gambiae P450s that metabolize pyrethroids and other insecticides. To elucidate catalytic sites
in some of the Papilio and Helicoverpa CYP6B proteins,
we have constructed substrate-docked models of the
CYP6B1 and CYP6B8 proteins based on sequence alignments with bacterial CYP102. In the CYP6B1 model, multiple aromatic amino acids contribute to the aromatic-aromatic network that stabilizes this P450’s catalytic site and
allows for interactions with this P450’s hydrophobic furanocoumarin substrates. Mutational analyses coupled with
baculovirus expression studies support these roles in substrate recognition. In the CYP6B8 model, many of these
aromatic residues are replaced with residues allowing for
the formation of a more open catalytic pocket capable of
accepting furanocoumarins as well as a range of plant allelochemicals and insecticides. Substrate binding analyses in
a number of CYP6B1 catalytic site mutants have indicated
that the altered activities for many of these mutants correlate with changes in heme spin state effected by alterations
in the hydrogen bonding network surrounding the heme.
S96
Additional modeling and mutagenesis in the CYP6B8 catalytic site is focused on identifying residues interacting with
the broader range of substrates metabolized by this P450.
MP41 P450-DEPENDENT ALKANE
MONOOXYGENASE OF
ACINETOBACTER SP. EB104
IS ENCODED IN A COMPOSITE
TRANSPOSON OF THE PLASMID
PAC450
MP40 TRANSCRIPT PROFILING OF
ARABIDOPSIS THALIANA P450S
SHAHJAHAN ALIa, HUI DUANa,
YURDAGUL FERHATOGLUa, MARK BANDb,
DANIELE WERCK-REICHHARTc, and
MARY SCHULERa
MARIA LUDEWIGa, CARSTEN SCHWARZb,
OTMAR ASPERGERa, SVANTE PÄÄBOb, and
ULRICH HAHNc
a
University of Leipzig, Institute of Biochemistry, Brüderstr.
34, 04103 Leipzig, Germany, bMax-Planck-Institute for Evolutionary Anthropology, Deutscher Platz, 04103 Leipzig,
Germany; cUniversity of Hamburg, Department of Bio-chemistry and Molecular Biology, Martin-Luther-King-Platz 6,
20146 Hamburg, Germany, [email protected]
a
Department of Cell and Structural Biology and bW.M.
Keck Center for Comparative and Functional Genomics,
University of Illinois, 1201 West Gregory Drive, Urbana,
IL61801, USA, cInstitute of Plant Molecular Biology,
CNRS, Strasbourg, France
In addition to the P450-independent alkane monooxygenase found in many strains of the genus Acinetobacter,
the species EB104 – that is able to grow on the toxic
middle-chain n-alkanes - contains an alkane monooxygenase consisting of the P450non (CYP153), the ferredoxin
nonadoxin (Nd) and the NADH-dependent nonadoxin reductase1. With the aim to exploit this unusual bacterial alkane monooxygenase as a biocatalyst and model enzyme
for the biotransformation of chemically highly inert and
very hydrophobic compounds its genetics were studied in
more detail. Southern hybridisation experiments2 show
that the three genes of the alkane monooxygenase which
are organised in a single operon3 are localised together
with the gene of a putative transcription regulator on the
native plasmid pAC450 of Acinetobacter sp. EB104. The
regulator protein belongs to the family AraC/XylS. Sequencing of the entire plasmid revealed that the P450 regulon is encoded within a 17-kb composite class-I transposon suggesting the acquirement of this metabolic feature
by means of horizontal gene transfer. Putative genes of
further enzymes of alkane degradation could not be detected on the plasmid, neither within the transposon nor outside of it. The transposon is flanked by two identical IS
elements each containing a transposase with 99% nucleotide sequence identity to the transposase in the transposon
Tn5047 of plasmid pKLH201 from Acinetobacter calcoaceticus KHW144. Tn5047 carries mercury resistance the
genes of which have not been detected in plasmid pAC450
of A. sp. EB104. Comparable high similarities could not
be found for any other putative ORFs detected on the plasmid. Further sequence analysis is in progress to get still
more information on origin and relationship of the
pAC450 components and its importance for the bacterium
A. sp. EB104. Altogether, the results described here support the hypothesis that the acquirement of the P450non
system by means of the plasmid pAC450 enables this bacterium to withstand toxic environment by conferring resi-
In plants, P450s function in the biosynthesis of lignins,
pigments, defense compounds, fatty acids, hormones and
growth regulators as well as in the metabolism of herbicides, insecticides and pollutants. Towards defining the function of the 272 P450 genes existing in Arabidopsis, we
have analyzed P450 gene-specific microarrays for constitutive and inducible transcript profiles in seedlings exposed to a variety of chemicals and environmental stresses.
Among several chemicals analyzed for their ability to induce transcription of sets of P450 genes are several plant
signaling molecules (JA, SA), phenobarbital, herbicides
(DICAMBA, atrazine) and fungal defense activators
(BTH). Among the stresses analyzed so far are osmotic and
cold stresses. Expression profiles for subsets of these
P450s have been correlated with RT-PCR analyses using
gene-specific probes and with oligonucleotide array profiles. These analyses indicate that some subsets of these
P450s are expressed in response to specific chemical cues
while other subsets of these P450s are expressed in response to overlapping sets of chemical cues. Heterologous
expression of several Arabidopsis P450s in baculovirus expression systems coupled with substrate binding analyses
is being used to define the ranges of chemicals hydroxylated by each of these sequences.
S97
stance against middle-chain alkanes and other dangerous
hydrocarbons, respectively.
accordance with the data in litterature. However, if it is
reported that CYP2C19 oxygenates arachidonic acid to
19-HETE, 14,15-EET and 8,9-EET which are vasoactive
compounds, the low catalytic activity of CYP2C19*2 do
not contribute to the changes in blood pressure.
REFERENCES
1. Asperger, O., Kleber, H. P. in: The Biology of Acinetobacter, (Towner, K. J., et al. eds.), pp. 323, Plenum
Press, New York and London 1991.
2. Galluhn, D.: Diploma thesis, University of Leiozig
1999.
3. Maier, T., Förster, H. H., Asperger, O. Hahn, U. Biochem. Biophys. Res. Commun. 286, 652 (2001).
4. Kholodii, G. Y.: EMBL AJ251307.2
REFERENCES
1. De Morais S.M., Wilkinson G.R., Blaisdell J., Nakamura K., Meyer U.A., Goldstein J.A. : J Biol Chem 269,
15419 (1994).
2. Bylund J., Ericsson J., Oliw E.H. : Anal Biochem. 265,
55 (1998).
3. Ibeanu G.C., Goldstein J.A., Meyer U., Benhamou S.,
Bouchardy C., Dayer P., Ghanayem B.I., Blaisdell J. :
J. Pharmacol. Exp. Ther. 286, 1490 (1998).
MP42 CYP2C19 DO NOT CONTRIBUTE
TO BLOOD PRESSURE.
MP43 COMPARISON OF NADPH-,
NAD+- AND UDP-GLYCOSIDEDEPENDENT BIOTRANSFORMATION OF BOHEMINE AND
ROSCOVITINE IN VITRO
C. BERTRAND-THIEBAULTa, B. MARIEa,
S. DROECHa, L. FERRARIa, A. THOMPSONb,
D. FROENZLERb, S. VISVIKISa, and AM. BATTa
a
INSERM U525, 30 rue Lionnois, Université Henri Poincaré, 54000, Nancy, France, bRoche Genetics, F. Hoffmann-La Roche, Ltd, PRBG-T, Bau 93/4.26, CH-4070,
Basel, Switzerland.
K. âERVENKOVÁa, Z. CHMELAa, M. BELEJOVÁb,
L. UHERKOVÁa M. RYPKAa, and D. RIEGROVÁa
The polymorphism of CYP2C19 is due to at least two
major and several minor variant alleles of CYP2C19
among the 19 alleles reported (www.imm.ki.se/CYPalleles/cyp2c19.htm) (20.02.03). The principal defective allele
CYP2C19*2 consist of an aberrant splice site in exon 51.
CYP2C19 is usually characterized by S-mephenitoin
4’-hydroxylation or omeprazole 5-hydroxylation. In poor
metabolizer (PM), both activities are extremely low. PMs
are representing 2-5% of caucasians. CYP2C19 is also able
to oxygenate arachidonic acid to 19-HETE, 14,15-epoxyeicosatrienoic acid (EET) and 8,9-EET as main metabolites2. For this last reason, we investigated the possible linkage between blood pressure and the polymorphism of
CYP2C19 among the subjects included in the Stanislas Cohort. This study observed 545 unrelated adult Caucasians
enroled in the Stanislas Cohort. We used Kinetic Thermal
Cycler method to determine CYP2C19 genotype. The relationship between genotype and continuous variables was
assessed by one-way ANOVA. Multiple linear regression
analysis was performed to evaluate the association between
CYP2C9*2 polymorphism and blood pressure. The frequency of the genotype mutation for rare allele was 4.2% in
position 681 and 3.8% in position 990. The allele frequency of CYP2C19*2 990 C/T was 17.7% ± 1.2 and the frequency of CYP2C19*2 681 G/A was 18.2% ± 1.2. The
observed frequencies of the homozygotes (CC vs GG) and
heterozygotes (CT vs GA) genotypes were in agreement
with those predicted according to the Hardy-Weinberg rule.
The allele frequencies observed for CYP2C19 were in
a
Department of Pathological Physiology, Medical Faculty,
Palack˘ University, Olomouc, Czech Republic, bPharmacological Institute, Medical Faculty, Palack˘ University, Olomouc, Czech Republic, [email protected]
The purpose of the present study was to utilize subcellular fractions of mouse liver and kidney to compare biotransformation of 6-benzylamino-2-(3-hydroxypropylamino)-9-isopropylpurine (bohemine) and 6-benzylamino2-(1-hydroxy-methylpropylamino)-9-isopropylpurine (roscovitine).
Methods
The compounds were 3H-labeled on the C8 atom of purine ring. The following systems were used to evaluate the
biotranformation: 1. Microsomal liver fraction + NADPH;
2. Microsomal liver or kidney fraction + UDP- glucose or
UDP- glucuronic acid; and 3. Cytosolic liver or kidney
fraction + NAD+. Kinetic data were obtained by sampling
the incubation mixtures in intervals followed by their denaturation, thin-layer chromatography, and autoradiography with densitometric evaluation.
Results
First, roscovitine was transformed, by mouse liver microsomal NADPH-dependent system, about twice as rapidly
than bohemine. The main product resulting from roscovitine was its corresponding carboxylic acid while bohemine
S98
latta) were incubated in 95% O2/5% CO2 atmosphere. Incubation medium was sampled in regular intervals and metabolite production was followed up by TLC /3H autoradiography with densitometric evaluation.
was preferentially metabolized by dealkylation processes1.
Second, in the microsomal-UDP-glycoside systems, bohemine glucosidation was catalyzed about fifty times (liver)
and five times (kidney) faster than that of roscovitine. Furthermore, glucuronidation of bohemine was catalyzed, to
a limited extend, by the liver and, even less, by kidney microsomes while glucuronidation of roscovitine was not detected. Third, in both liver and kidney cytosolic, NAD+dependent systems, oxidation of bohemine into its corresponding carboxylic acid occured. Roscovitine was transformed about fifteen times more slowly than bohemine by
liver cytosol and no reaction of roscovitine occured in the
kidney cytosol.
Supported, in parts, by the Ministry of Education,
Youth and Sports of the Czech Republic (MSM
151100001, COST OC B17.20) and Grant Agency of the
Czech Republic (301/02/0475/A).
Results
All tested substances produced metabolites that began to
appear within the first 30 minutes of incubation. Appropriate carboxylic acids that arose by oxidation of a -CH2OH
side-chain group situated on C2 atom of the purine ring were
produced preferentially from the parent compounds. However, mouse kidney slices were unable to transform bohemine
into the appropriate acid and monkey liver and kidney slices
from all tested species were unable to transform roscovitine
into the appropriate acid. A.2.J.36 and A.2.3.9.(R) were only
slightly oxidized into their acids or not at all. Additionally,
bohemine was glucosylated by mouse kidney slices, producing bohemine-O-βD-glucoside, above all the other compounds. Other glycosylated metabolites - glucuronides were the main products created from A.2.J.36 and A.2.3.9
(R) by mouse, rat and monkey kidney slices. Except of
mouse and rat kidney slices in the presence of A.2.J.36, the
liver slices metabolized the tested substances more effectively than the kidney ones. Notably, roscovitine emerged as
the most stable parent compound among all.
Conclusions
Roscovitine exerted higher degree of metabolic stability over bohemine when explored in vitro. This is in
accord with the evidence of its slower clearance obtained
in in vivo experiments in mice2.
REFERENCES
Conclusions
These observations are in concordance with those received in in vitro studies of bohemine and roscovitine metabolism exploiting mouse subcellular fractions supplemented with NADPH, NAD+, and/or glycosyl donors1, and with
those of bohemine and roscovitine in in vivo experiments
in mice2. The work demonstrates how the precission-cut
tissue slices technique can complete data obtained using
other model systems.
1. Rypka M., Vesel˘ J., Chmela Z., Riegrová D., âervenková K., Havlíãek L., Lemr K., Hanu‰ J., âern˘ B., Luke‰ J., Michalíková K.: Xenobiotica 32,
2. Chmela Z. et al.: 13th Int. Conf. on Cytochromes P450,
Prague, June 29 - July 3, 2003, Abstr. M46
MP44 BIOTRANSFORMATION OF
OLOMOUCINE-TYPE
CYCLIN-DEPENDENT KINASE
INHIBITORS BY
PRECISSION-CUT TISSUE
SLICES FROM DIFFERENT
ANIMAL SPECIES
*)bohemine: 6-benzylamino-2-(3-hydroxypropylamino)-9isopropylpurine
roscovitine: 6-benzylamino-2-(1-hydroxymethylpropylamino)-9-isopropylpurine
olomoucin: 2-(hydroxyethylamino)-6-benzylamino-9methylpurine
K. âERVENKOVÁ, Z. CHMELA, and M. RYPKA
Compound A.2.J.36:
Department of Pathological Physiology, Medical Faculty,
Palack˘ University, Olomouc, Czech Republic
[email protected]
2-(2-hydroxy-1-isopropylethylamino)-6-(2-hydroxybenzylamino)-9-isopropylpurine
Compound A.2.3.9.(R): 2-(2-hydroxy-1-ethylamino)-6(3-hydroxybenzylamino)-9-isopropylpurine
The aim of this study was to compare biotransformation of 3H-labeled olomoucine, bohemine, roscovitine,
compound A.2.J.36, and compound A.2.3.9.(R)* by liver
and kidney slices of rodents and primates.
153110001, COST B5/B17) and Grant Agency of the
Methods
Liver and kidney slices (8 mm in diametr; thickness
200-250 µm) from mouse, rat and monkey (Macaca mu-
S99
REFERENCES:
1. âervenková K. et al.: 13th Int. Conf. on Cytochromes
P450, Prague June 29 - July 3, 2003, Abstr. M43
2. Chmela Z. et al.: 13th Int. Conf. on Cytochromes P450,
Prague June 29 - July 3, 2003, Abstr. M46
MP45 SIGNIFICANT ROLE OF
CYP2A AND CYP3A IN
BIOTRANSFORMATION OF
BOHEMINE BY MOUSE LIVER
MICROSOMES IN VITRO
Conclusions
The present findings will facilitate experiments designed to dissect enzymatic systems catalyzing clearence of
C2,C6,N9-trisubstituted purine compounds from mammalian tissues.
151100001, COST OC B17.20), and Grant Agency of the
REFERENCES
1. Rypka M., Vesel˘ J., Chmela Z., Riegrová D., âervenková K., Havlíãek L., Lemr K., Hanu‰ J., âern˘ B., Luke‰ J., Michalíková K.: Xenobiotica 32, 1017 (2002).
M. RYPKAa, Z. CHMELAa, K. âERVENKOVÁa,
K. LEMRb, and D. RIEGROVÁa
a
Department of Pathological Physiology, Medical Faculty, Palack˘ University, Olomouc, Czech Republic,
b
Department of Analytical Chemistry, Faculty of Natural
Science, Palack˘ University, Olomouc,
[email protected]
The aim of this study was to characterize the biotransformation of selective cyclin-dependent kinase inhibitor 6benzylamino-2-(3-hydroxy-propylamino)-9-isopropylpurine (bohemine) by mouse liver microsomes.
Methods
Metabolite profiles of 8-3H-labelled bohemine were
established by TLC/3H-autoradiography, and enzymatic and
MS analyses were utilized to elucidate chemical structures
of the metabolites. Participation of individual CYPs was followed up by testing with enzyme selective CYP inhibitors1.
Results
Scheme of primary NADPH-dependent pathways have
been proposed involving N2- and N9-dealkylation, N6-debenzylation, aromatic hydroxylation, and C2-side chain oxidation of bohemine. Three of the primary metabolites detected, namely 6-benzylamino-2-(3-hydroxypropylamino)purine (M-4), 6-amino-2-(3-hydroxypropylamino)-9-isopropylpurine (M-5), and 6-(4-hydroxybenzylamino)-2-(3-hydroxypropylamino)-9-isopropylpurine (M-6), all of them retaining their parent primary hydroxyl group, were subsequently shown to be converted by a liver cytosolic, NAD+-dependent system, into their corresponding carboxylic acids.
Metabolite M-6 was subject to microsomal glycosidations
requiring UDP-sugar donors. NADPH-dependent conversion of M-6 into M-5 by microsomes was also demonstrate
substrate (8-methoxypsoralen, ketoconazole, troleandomycin and coumarin) were utilized to identify CYPs involved
in bohemine biotransformation. The findings suggested that
CYP2a and CYP3a substantially contribute to NADPH-dependent bohemine transformation in vitro.
S100
MP46 CARBOXYLIC ACID IS THE
MAIN PRODUCT OF
ROSCOVITINE BIOTRANFORMATION IN MICE IN VIVO.
COMPARISON WITH
BOHEMINE
Z. CHMELAa, M. RYPKAa, K. âERVENKOVÁa,
K. LEMRb, D. RIEGROVÁa, and J. VESEL¯a
a
Department of Pathological Physiology, Medical Faculty, Palack˘ University, Olomouc, Czech Republic,
b
Department of Analytical Chemistry, Faculty of Natural
Science, Palack˘ University, Olomouc
[email protected],
The purpose of the present study was to investigate the
biotransformation of selective cyclin-dependent kinase inhibitor 6-benzylamino-2-(1-hydroxymethylpropylamino)9-isopropylpurine (roscovitine) in mice in vivo and to
compare it with that of 6-benzylamino-2-(3-hydroxypropylamino)-9-isopropylpurine (bohemine) in vivo1.
Methods
3
H-labeled parent compounds were administered intravenously and 3H-radioactivity in blood, liver, kidney, urine,
and gut was monitored by liquid scintillation counting.
Levels of the parent compounds and those of their metabolites were assessed by thin-layer chromatography and
autoradiography1, 2.
Results
The level of roscovitine in blood decreased from 5.9 %
of the administered dose (2 min after the administration) to
0.7 % (30 min after the administration). The level of bohemine declined more rapidly (from 4.6 % in 2 min to 0.15 %
in 30 min)1. The liver and kidney were the organs responsible for biotransformation and elimination of both com-
pounds. A carboxylic acid was produced from roscovitine
by oxidation of its -CH2OH side group. The carboxylic acid
was the main metabolite of the parent compound. Based on
the additional findings obtained in vitro, this oxidation
mostly ocured in NADPH-dependent manner3. On the other
hand, bohemine carboxylic acid was also identified.
However, its formation was mainly mediated by cytosolic,
NAD+-dependent, 4-methylpyrazol sensitive alcohol dehydrogenase class I1.
Conclusions
The clearance of roscovitine in mice is about four times
slower than that of bohemine. The result is of direct practical importance considering a preclinical testing of these
compounds as potential novel anticancer agents.
151100001, COST OC B17.20), and Grant Agency of the
Recombinant enzymes of rat CYP2D1, CYP2D2,
CYP2D3 and CYP2D4, and human CYP2D6 were expressed in yeast2. Each microsomal fraction was incubated with
progesterone and 17OH-progesterone, and those 21-hydroxylated metabolites were quantified by LC-MS/MS.
Only rat CYP2D4 and human CYP2D6, predominant
CYP2D isoforms in the brain, possessed 21-hydroxylation
activities for progesterone and 17OH-progesterone. These
activities were also observed in rat brain microsomes and
could be effectively inhibited by quinidine and antiCYP2D antibodies. RT-PCR and Western blot analysis revealed that mRNA and protein of CYP2D4 distributed
throughout the rat brain whereas those of P450c21 were solely detected. Furthermore, rat CYP2D4 and human
CYP2D6 catalyzed 21-hydroxylation of allopregnanolone,
one of the representative allosteric GABA modulator.
These results strongly suggest that the brain CYP2D isoforms play an important role in the regulation of brain neurosteroids level as steroid 21-hydroxylase.
REFERENCES
REFERENCES
1. Chmela Z., Vesel˘ J., Lemr K., Rypka M., Hanu‰ J.,
Havlíãek L., Kry‰tof V., Michnová L., Fuksová K., Luke‰ J.: Drug. Metab. Dispos. 29, 326 (2001).
2. Rypka M., Vesel˘ J., Chmela Z., Riegrová D., âervenková K., Havlíãek L., Lemr K., Hanu‰ J., âern˘ B.,
Luke‰ J., Michalíková K.: Xenobiotica 32, 1017
(2002).
3. âervenková K. et al.: 13th Int. Conf. on Cytochromes
P450, Prague, June 29 - July 3, 2003, Abstr. M43.
1. Hiroi T., Kishimoto W., Chow T., Imaoka S., Igarashi
T., Funae Y.: Endocrinology 142, 3901 (2001).
2. Wan J., Imaoka S., Chow T., Hiroi T., Yabusaki Y., Funae Y.: Arch. Biochem. Biophys. 348, 383 (1997).
MP47 CYP2D IS INVOLVED IN THE
NEUROSTEROIDOGENESIS IN
THE BRAIN
MP48 ANALYSIS OF CYTOCHROME
P450S IN A PYRETHROID
RESISTANT STRAIN OF
HELICOVERPA ARMIGERA
(COTTON BOLLWORM)
VLADIMIR GRUBOR, and DAVID G. HECKEL
Centre for Environmental Stress and Adaptation Research,
Department of Genetics, University of Melbourne, Parkville, VIC 3010, AUSTRALIA
[email protected]
WATARU KISHIMOTOa,b, TOYOKO HIROIa,
MAYUKO OSADAa, SUSUMU IMAOKAa,
TAKASHI IGARASHIb, and YOSHIHIKO FUNAEa
a
Department of Chemical Biology, Osaka City University
Medical School, Osaka 545-8585, Japan, bDepartment of
Drug Metabolism and Pharmacokinetics, Kawanishi Pharma
Research Institute, Nippon Boehringer Ingelheim Co., Ltd.,
Hyogo 666-0193, Japan, [email protected]
We previously reported that the brain CYP2D4 and
CYP2D6 are involved in progesterone 21-hydroxylation1.
In the brain, the enzyme responsible for steroid 21-hydroxylation has not been well elucidated although very small
amount of P450c21 mRNA was present. In this study, we
investigated whether CYP2D functions as steroid 21-hydroxylase in the brain. We also focused our efforts on the regional CYP2D expression in the brain using RT-PCR and
Western blot analysis.
S101
Helicoverpa armigera is a serious pest of cotton and other crops around the world. It has evolved resistance to
most classes of insecticides. The AN02 strain from Australia exhibits 50 fold larval resistance and 100 fold adult resistance to the pyrethroid, fenvalerate. This resistance is
suppressible by piperonyl butoxide (suggesting a detoxicative mechanism), and past work has focused on the
Cyp6B family of cytochrome p450s. Linkage mapping
with AFLPs was used to assign the fenvalerate resistance
gene to Linkage Group 13 (LG13), while a Cyp6B cluster
maps to LG14. This argues against Cyp6B cis-acting factors affecting resistance, but would be consistent with
trans-acting factors on LG13 affecting Cyp6B expression
levels. A larval midgut cDNA library was screened for
p450 genes and several members of the Cyp9A family
were recovered, but not Cyp6B. The most abundant
Cyp9A p450 was mapped to LG10.
Real-time RT-PCR was used on individuals from both
resistant and susceptible strains to explore tissue specific
and developmental expression of CYP6s and CYP9s. Preliminary results suggest there is individual variation in relative expression levels of Cyp6B2, Cyp6B6, and Cyp6B7
that may have a genetic basis.
MP49 INFLUENCE OF RECIPIENT
GENDER ON CYTOCHROME
P450 ISOFORMS EXPRESSION
AND ACTIVITY IN
INTRASPLENIC FETAL LIVER
TISSUE TRANSPLANTS IN
COMPARISON TO LIVER
IN RATS
AMELIE LUPP, SABINE HUGENSCHMIDT, and
DIETER MÜLLER
Institute of Pharmacology and Toxicology, Friedrich Schiller University, Nonnenplan 4, D-07743 Jena, Germany;
[email protected]
Ectopic liver cell transplants can serve as a tool to
study humoral, nerval and other topic influences on liver
cell differentiation, multiplication, function and sensitivity
to foreign compounds. Rat livers display a sex-specific cytochrome P450 (P450) isoforms expression pattern which
is regulated by a differential profile of growth hormone
(GH) secretion in male and female rats. The aim of the present study was to elucidate whether intrasplenic fetal liver
tissue transplants of originally mixed sex are subject to the
same regulatory influence of the GH secretion pattern as
the cells within the orthotopic livers and thus will adapt to
the gender of the recipients with respect to P450 isoforms
expression.
Syngenic fetal liver tissue suspensions of mixed sex
were transplanted into the spleens of adult male or female
Fischer 344 rats. Four months after surgery transplant-recipients and age-matched control animals were treated either
with (-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) or the solvent and sacrificed 24 or 48
hours thereafter. Expression of the P450 isoforms 1A1,
2B1, 2E1, 3A2 and 4A1 in livers and transplant-containing
spleens was assessed by immunohistochemistry. P450 dependent monooxygenase functions were measured biochemically using different model reactions for distinct P450
subtypes: ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), p-nitrophenol hydroxylation (PNPH), pentoxyresorufin O-depentylation (PROD)
and testosterone hydroxylation (TH) at different positions.
S102
The livers of both male and female rats displayed nearly no P450 1A1, but a distinct P450 2B1, 2E1, 3A2 and
4A1 expression. Whereas no gender differences were observed in the immunostaining for P450 1A1, the expression
of P450 2B1, 3A2 and 4A1 was stronger in males and that
for P450 2E1 in females. Like in the livers the immunostaining for P450 2B1, 3A2 and 4A1 was more pronounced in
transplants of male recipients and that for P450 2E1 in
those of female hosts. With some sex-specific differences
both in livers and in transplants the P450 1A1 and 2E1 expression was increased by BNF, that of P450 2B1 by PB,
and that of P450 3A2 by DEX. Spleens of control rats displayed nearly no P450 isoforms expression. Additionally,
only very low monooxygenase activities were detectable in
the spleens of male or female control rats. In contrast, with
most model reactions distinct activities were seen in the
transplant-containing organs. Like the respective livers,
transplant-containing spleens from male rats displayed higher basal ECOD and 2α-, 2β-, 6β-, 14α-, 15α-, 15β-, 16α-,
16(- and 17-TH activities than those from females, whereas
the opposite was the case for the PNPH and 6α- and 7α-TH
activities. No gender dependence was seen with basal
EROD, PROD and 15β-TH activities. With nearly all model reactions the sex-specific differences in the inducibility
by BNF, PB or DEX seen in the livers could be observed
also in the transplant-containing spleens. P450 activities,
respective gender-differences and sex-specific inducibility
correlated well to the corresponding P450 enzyme expression patterns.
In summary, the results of the present investigation indicate that the P450 system of the intrasplenically transplanted liver cells is influenced by the recipient gender like
that of the livers.
MP50 HIGH-THROUGHPUT
LC-MS/MS METHOD FOR
CYP2D6 PHENOTYPING WITH
DEXTROMETHORPHAN
THOMAS E. MÜRDTERa, GERHARD SCHÄNZLEb,
GEORG HEINKELEa, KARIN ENDRIZZIa,
CLAUDIA MARXa, MATTHIAS SCHWABa,
MICHEL EICHELBAUMa, and ULI ZANGERa
a
Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology D-70376 Stuttgart, Germany, bInstitute of Pharmacokinetics and Metabolism, Merck KGaA, D-85567
Grafing, Germany, [email protected]
Background
In contrast to methods for genotyping, phenotyping
usually still needs time consuming sample preparation and
analytical procedures. On the other hand, the transformation of a probe drug gives a better understanding of the actual metabolic situation. Therefore, we developed a high-
through put method for the determination of dextromethorphan and its phase I and phase II metabolites.
Methods
Healthy volunteers (n=31) received 30 mg dextromethorphan once monthly followed by a 12 hours urine collection period over a time period of 7 months. Aliquots of
the urine were spiked with a mix of stable isotope labelled
internal standards and 10 µl were injected on the LCMS/MS system with on line sample preparation (API 3000,
Applied Biosystems). Total cycle time was 6 min. Genotype of CYP2D6 was determined by multiplex PCR.
Results
Dextromethorphan (DT), dextrorphan (DX), dextrorphan glucuronide (DXG), dextrorphan sulfate (DXS), methoxymorphinan (MM), 3-hydroxymorphinan (HM) and
morphinan glucuronide (MG) could be quantified within
a single chromatogram in less than 4 min. With limits of
quantification ranging from 4 to 37nM MG could be quantified even in PMs. The metabolic ratio (MR) calculated
from DX + DXG + DXS and DT correlated with the MR
calculated from HM + MG and MM (r2=0.89, p<0.0001).
Both logMR showed a trimodal distribution with separate
subgroups for PMs, extensive metabolizers (EMs) and intermediate metabolizers. In contrarst, both logMR accounting for glucuronidation and sulfation showed a normal distribution although PMs had lower glucuronidation and
sulfation rates compared to EMs (p<0.0001).
Conclusion
The described method is suitable for phenotyping for
CYP2D6 and can give additional information about glucuronidation and sulfation capacities.
Supported by the Robert Bosch Foundation, Stuttgart.
MP51 INFLUENCE OF METHAMPHETAMINE ON ACTIVITY OF CYP
2D1 IN THE ISOLATED
PERFUSED WISTAR ALBINO
RAT LIVERS
JI¤Í SLÍVA
Masaryk University, Faculty of Medicine, Department of
Pharmacology, Brno, Czech Republic
It is known that methamphetamine influences the oxidative metabolism in the liver (in the pathway of cytochrome
P450 2D1). The present study used the model of isolated
perfused liver of Wistar albino rats to ascertain the influence
of methamphetamine on cytochrome P450 2D1 based on
analysis of the metabolites of dextromethorphane (DEM).
DEM is a typical substrate for that cytochrome isoenzyme.
S103
For treatment twenty four rats were randomly divided into
3 groups: a) 2 ml of saline solution/kg/day for 10 days, intraperitoneally, b) methamphetamine 10mg/kg/day for 10 days,
intraperitoneally c) methamphetamine 10 mg/l in vitro. The
quantitative evaluation of the DEM metabolites was performed by HPLC (high pressure of liquide chromatography). For the statistical analysis of the results the Student t- test was used. The significant difference comparing
to controls was found just in the concentration of the main
DEM metabolite dextrorphane in the last period of perfusion (after 120 min) in the group of rats pretreated 10 days
in vivo with methamphetamine, 10 mg/kg/ day. This finding indicates the stimulation of cytochrome 2D1. Furthermore, a small trend of insignificant increase in the amount
of some other metabolites was recorded. Thus, the present
results brought evidence on possible facilitative influence
of MET on the activity of CYP 2D1.
MP52 EXPRESSION OF ARYL
HYDROCARBON RECEPTOR
REPRESSOR IN NORMAL
HUMAN TISSUES AND
INDUCIBILITY BY POLYCYCLIC
AROMATIC HYDROCARBONS
IN HUMAN TUMOR-DERIVED
CELL LINES
YUKI TSUCHIYA, MIKI NAKAJIMA, SATSUKI ITOH,
MASASHI IWANARI, and TSUYOSHI YOKOI
Division of Drug Metabolism, Faculty of Pharmaceutical
Sciences, Kanazawa University, Takara-machi 13-1, Kanazawa 920-0934, Japan
[email protected]
Aryl hydrocarbon receptor repressor (AhRR) has been
recently identified as a negative factor that suppresses aryl
hydrocarbon receptor (AhR)-mediated transcriptional gene
expression. In the present study, the expression level of
AhRR in normal human tissues was determined. AhRR
mRNA was detected in liver, breast, colon, kidney, lung,
bladder, uterus, testis, ovary, and adrenal gland. The expression level in the testis was prominently high. AhRR
mRNA was also detected in various human tissue-derived
cell lines, HepG2 (hepatocellular carcinoma), MCF-7 (breast carcinoma), LS-180 (colon carcinoma), ACHN (renal
carcinoma), A549 (lung carcinoma), HT-1197 (bladder carcinoma), HeLa (cervix of uterus adenocarcinoma), NEC14
(testis embryonal carcinoma), and OMC-3 (ovarian carcinoma). Since the expression level of AhRR mRNA was
prominently high in HeLa cells, it is suggested that the
high expression level of AhRR might work as a negative
factor for the low inducibility of the CYP1 family in HeLa
cells. The expression of AhRR mRNA was induced by
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3-methylchoranthrene (3-MC) in HepG2, MCF-7, LS-180, and
OMC-3 cells, but not in ACHN, A549, HT-1197, HeLa,
and NEC14 cells. The responsiveness was similar to the
cell-specific inducibility of the CYP1 family. The inducibility of AhRR mRNA by nitropolycyclic aromatic hydrocarbons (NPAHs) as well as their parent PAHs was compared in HepG2 and OMC-3 cells. The chemical-specific inducibility of AhRR was also similar to that of the CYP1 family determined in our previous study. These results indicated that AhRR is also induced in chemical- and cell-specific manners.
MP53 REGULATION OF MUTATION
PROCESS INDUCED BY
IONIZING RADIATION IN
MYELOCARIOCYTES OF MICE
BY PEROXIDASE AND
EXTRACT FROM
ARMORACIA RUSTICANA L.
RENA AGABEYLIa, TUBU GASIMOVAa, and
URKHAN ALEKPEROVb
a
Institute of Botany, Patamdartskoe shosse 40, 370073 and
Baku State Universiti, Baku, Azerbaijan Republic, bWestern University, Baku, Azerbaijan Republic
urkhan. alekperov@ un. azeri. com
The participation of antioxidant enzymes in a regulation of mutation and aging processes induced by environmental genotoxicants has been shown in series of works1-7.
The special actuality is acquired study of plant extracts as
perspective agents for prevention both spontaneous and induced mutation process. The genetic activity of peroxidase
- EC 1.11.1.7 (P) (Reanal) and extract (EHR) from a roots
1
2
3
4
5
6
-
control
EHR
P
γ-R
EHR + γ-R
P + γ-R
Fig. 1. The influence of (P) and EHR on frequency of spontaneous
and induced by (GR) chromosome aberrations in marrow cells of
mice.
S104
of a horse-radish (Armoracia rusticana L.) and their influence on frequency of spontaneous and induced by gamma
rays aberrations of chromosomes (CA) in myelocariocytes
of mice have been studied. The males of laboratory mice,
2-month age and with mass 18-20g have been used as object of experiments. Method - the analysis of CA frequency
in mice marrow cells. The P and EHR administrated per os
(30mg/kg) daily during 6 days before irradiation. Then,
animals irradiated by gamma-rays (GR) in 1Gr dose,
20 rad/sek, and source is Co60 and mortified in 24 hours
after irradiation. The mice divided into 6 groups, on 5 in
each group: 1) control, standard ration (SR); 2) SR+P;
3) SR+EHR; 4) SR + GR; 5) P+GR; 6) EHR+GR. The administration to the SR of P and EHR has not increased the
control level of CA in marrow cells of mice (Fig. 1). In experiments have been shown the tendency of the CA frequency decreasing in these variants. The addition to SR of
P completely neutralizes genotoxic action of GR and decrease frequency the of CA from 10.68±1.68 % to
1.54±0.39 %, P < 0.001; EHR till 4.31±0.59, P < 0.001.
The comparative assessment of the antimutagenic efficiency of EHR and P has revealed high efficiency of P
(85 %), the efficiency of EHR has made 54 %. The experiments had shown the ability of P and EHR to prevent the
mutation process, induced by GR in myelocariocytes of
mice. It is obvious that P as an element of nonspecific system can play a stabilizing role at various pathological processes, including, connected with a damage of the genetic
structures by ionizing radiations.
REFERENCES
1. Morita K., Hara M., Kada T.: Agr. Biol. Chem. 42, 1235
(1978).
2. Golubkova T., Gapanenko E., Pshenichnov R. et al.:
Bull. Gen. Soc., 25, suppl. 1, 31 (1994).
3. Agabeyli R.A.: ISSX Proceedings. 11. 6th European
ISSX Meeting Gothenburg, Sweden, 237 (1997).
4. Agabeyli R.A., Iskenderova I. M.: Drug Metabol. Rev.,
33, 461, 232 (2001).
5. Wall M.E.,Wani M.C., Huges T.L. et al.: Antimutagenesis and Anticarcinogenesis Mechanisms (II), New York,
London, p. 61 (1990).
6. Mitscher L.,Telikepalli N., Mc. Ghee E. et al.: Mut.
Res., 305, 143 (1996).
7. Alekperov U.K. Eur. J. Canc., 11 Suppl. 2., 8 (2002).
MP54 MOUSE CYTOCHROMES P450 4F
MP55 TCDD INDUCES CYP2A5
ENZYME IN MOUSE PRIMARY
HEPATOCYTES
LEPOSAVA ANTONOVIC, XIAOMING CUI,
HIDENORI KAWASHIMA, and HENRY W. STROBEL
Department of Biochemistry and Molecular Biology, The
University of Texas-Houston Medical School, P.O. Box
20708, 6431 Fannin Street, 77225, Houston, TX, USA
[email protected]
Cytochromes P450 4F (CYP 4F) have been newly discovered and are a subfamily of cytochromes P450 that
have hydroxylation activity toward many endogenous
compounds, such as fatty acids, prostaglandins and leukotrienes, which are important mediators involved in the inflammatory response. These findings suggest that CYP 4Fs
can modulate the level of these physiologically important
substances and in that way regulate the level of inflammation. Also inflammation itself may regulate the level of
CYP 4F expression and enzyme activity, which will further
affect the metabolism by CYP 4Fs. To date six family
members have been cloned in human and rodent. Four rat
isoforms have been cloned in our laboratory. Five novel
mouse cytochromes P450 4Fs genes, two from liver CYP
4F14 and CYP 4F15 and three from kidney CYP 4F13,
CYP 4F16 and CYP 4F18 were cloned in our laboratory.
The mouse isoforms share 90 percent or higher nucleotide
sequence homology with their orthologues in rat. Unlike
members of cytochrome P450 4A subfamily, expression of
CYP 4F isoforms is repressed by clofibrate in the rat liver
in contrast to CYP 4As, whose expression is induced by
clofibrate treatment. Rat CYP 4F isoforms also metabolize
exogenous drug substrates, the brain acting drugs, such as
imipramine and chlorpromazine. This work is focusing on
the study of mouse CYP 4Fs enzymatic activity, substrate
specificity, molecular basis of their regulation and their
role in inflammation.
REFERENCES
Kawashima, H., et all. J. Biol. Chem. 271, 28176 (1996)
Cui, X., et all. J. Pharmacol. Exp. Ther. 296, 547 (2001)
Kikuta, Y., et all. J Biol Chem. 268, 9376 (1993).
Kawashima, H., and Strobel, H. W., Biochem Biophys
Res Commun. 217, 1137 (1995)
5. Kawashima, H., et all. Arch Biochem Biophys. 347,
148 (1997)
6. Kikuta, Y., et all. J. Bioch. 127, 1047 (2000)
7. Boehme, C., and Strobel, H. W., Neurotox. Res. 00, 1
(2000)
1.
2.
3.
4.
S105
SATU ARPIAINEN, OLAVI PELKONEN, and
JUKKA HAKKOLA
of Oulu, Box 5000, FIN-90014 Oulu, Finland
[email protected]
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes several biochemical and toxic effects, such as teratogenesis,
immunosuppression and tumor promotion. It is, together
with other polycyclic aromatic hydrocarbons (PAHs),
a well-known inducer of xenobiotic metabolizing cytochrome P450 (CYP) enzymes of family 1, i.e. CYP1A1 and
CYP1B1, and aryl hydrocarbon receptor (AHR) mediated
mechanism is responsible for the induction1.
Mouse CYP2A5 and its human orthologue CYP2A6
metabolize many toxic substrates, such as nicotine, nitrosamines and aflatoxins. CYP2A5 is induced by number of
structurally diverse compounds including phenobarbital
and pyrazol2. Interestingly, we have discovered that also
TCDD induces CYP2A5. In the present study the mechanism of TCDD induction of CYP2A5 was investigated in
mouse primary hepatocytes.
1 µM TCDD caused 3-fold elevation of coumarin hydroxylation (COH) catalysed by CYP2A5 after 24 h exposure. Further, this was accompanied by similar elevation of
the CYP2A5 apoprotein level studied by immunoblotting.
Also CYP2A5 mRNA level was induced 2-3 fold by
TCDD treatment. -3033 to +10 of the Cyp2a5 5’-flanking
region was cloned in front of a luciferase reporter gene and
the construct was transfected into primary hepatocytes.
TCDD treatment induced luciferase activity 3-5 fold suggesting involvement of transcriptional regulation.
Regulation of gene expression by TCDD is predominantly mediated by AHR and its dimerization partner aryl
hydrocarbon nuclear translocator (ARNT). DBA/2 and
C56BL/6 mouse strains show genetically-determined differential responses to AHR ligands3. The induction of
Cyp2a5 by TCDD was studied in hepatocytes of these two
mouse strains and was found to correlate with Cyp1a1 induction suggesting involvement of AHR also in the induction mechanism of Cyp2a5.
REFERENCES:
1. Hankinson O: Annu Rev Pharmacol Toxicol 35, 307,
(1995).
2. Donato MT, Viitala P, Rodriguez-Antona C, Lindfors A,
Castell JV, Raunio H, Gomez-Lechon MJ, Pelkonen O:
Drug Metab Dispos 28, 1321, (2000).
3. Poland AP, Glover E, Robinson JR, Nebert DW: J Biol
Chem 249, 5599, (1974).
REFERENCES
MP56 CYP2C8, CYP2C9, CYP3A7, PXR
AND CAR MRNA LEVEL
INCREASE IN HEPATOBLASTOMA CELL LINE HEPG2, AFTER
HMG-COA REDUCTASE
INHIBITORS TREATMENT
C. BERTRAND-THIEBAULT, L. FERRARI,
C. MASSON, S.VISVIKIS, and AM. BATT
INSERM U 525, 30 rue Lionnois, 54000 NANCY, France,
[email protected]
Statins which are 3-hydroxy-3-methylglutarylcoenzyme A (HMGCoA) reductase inhibitors are largely used
to decrease plasma cholesterol level and reduce cardiovascular and cerebrovascular events in patients with established coronary artery disease. The phase I metabolism of
most statins involves the P450 subfamilies CYP3A and
CYP 2C. It was also demonstrated with lovastatin, that
these drugs, except pravastatin, activate the Pregnane
X Receptor (PXR)1 and up-regulate CYP3A1/2 in primary
cultured rat hepatocytes2. Fluvastatin and cerivastatin
increase CYP2C protein and mRNA expression in porcine
coronary artery endothelial cells3. CYP2C9 expression is
regulated by both nuclear receptors PXR and CAR, an orphan receptor4. This study focused on the effects of five
statins (atorvastatin, fluvastatin, lovastatin, pravastatin and
cerivastatin) on CYP2C8, CYP2C9, CYP3A7, PXR and
CAR mRNA expression in HepG2 hepatoblastoma cell
line. After 72 h treatment, atorvastatin, fluvastatin, lovastatin (0.1 to 10 µM) and cerivastatin (0.1 to 10 nM) increased CYP3A7 mRNA level (2-4 fold.). CYP2C8 and
CYP2C9 mRNA level increased 1.5-2.5 fold. After
96 h treatment, atorvastatin, fluvastatin, lovastatin (1mM)
clearly increased CYP2C9 protein level. Pravastatin (hydrophilic) had no effect on any CYP mRNA level tested.
After 48 h treatment, fluvastatin and cerivastatin increased
CAR mRNA expression (2.5 and 1.5 fold, respectively). In
the same conditions, fluvastatin increased PXR mRNA level. Atorvastatin increased PXR mRNA expression
(3.5 fold) after 72 h treatment. These increases in both
PXR and CAR nuclear receptors expression are related
with the increase in CYP2C9 mRNA expression. Statins, at
least lovastatin, are known to activate PXR receptor. Moreover, our data demonstrate that fluvastatin or cerivastatin
not only activate PXR, but also enhance PXR mRNA
expression, contributing in the increase in CYP3A7. All together, our data demonstrate that CYP2C8, CYP2C9 and
CYP3A7 and nuclear receptors PXR and CAR mRNA
expression are modulated by various lipophilic statins, suggesting that the increase in CYP3A and CYP2C mRNA
and protein expressions after treatment with these lipophilic statins results from their effects on both expression level and activation of nuclear receptors PXR and CAR.
S106
1. Kliewer S.A., Moore J.T., Wade L., Staudinger J.L.,
Watson M.A., Jones S.A., McKee D.D., Oliver B.B.,
Willson T.M., Zetterstrom R.H., Perlmann T., Lehmann
J.M.: Cell 92, 73 (1998).
2. Kocarek T.A. and Reddy A.B.: Drug. Metab. Dispos.
24, 1197 (1996).
3. Fisslthaler B, Michaelis UR, Randriamboavonjy V, Busse R, Fleming I.: Biochim. Biophys. Acta. 1619, 332
(2003).
4. Pascussi J.M., Gerbal-Chaloin S., Drocourt L., Maurel
P., Vilarem M.J..: Biochim. Biophys. Acta. 1619, 243
(2003).
MP57 CHARACTERIZATION OF
CYTOCHROME P450
EXPRESSION AND INDUCTION
PROFILES IN THE WIF-B9 CELL
LINE
FRANÇOISE BORDEa, VIRGINIE E. BENDERa,
STÉPHANE CHEVALIERa, CLAUDE CHARUELa,
DORIS CASSIOb, and CHRISTINE P. BIAGINIa
a
PFIZER, Molecular and Cellular Toxicology Laboratory,
Z.I. Pocé sur Cisse, 37401 Amboise, France, bINSERM
442, Université Paris-Sud, Bat. 443, 91405 Orsay, France
[email protected]
The WIF-B9 model is a unique, highly differentiated
and polarized hepatoma cell line, obtained by hybridization
of rat hepatoma cells (Fao) and WI38 human fibroblasts. It
presents morphological features, such as functional bile canaliculi1, that are close to those of primary hepatocytes2.
We previously demonstrated that WIF-B9 is a valuable
model for in vitro hepatotoxicity investigations3. We further evaluated the metabolic capacity of WIF-B9 using
competitive RT-PCR. Five rat isoforms (CYP 1A1, 2B1/2,
2E1 and 4A1) and four human isoforms (CYP 1A1, 2Cs,
2D6, 2E1) are constitutively expressed in this cell line. Human forms are expressed at 2-4 orders of magnitude lower
(ranking from 4 102 to 2.5 104 copies/ng total RNA) than
that of the rat forms (ranking from 4 105 to 6.4 106 copies/ng total RNA). The induction potential of these rat and
human P450 forms was assessed using seven different reference inducers. Rat CYP 1A1/2, 3A1/2 and 4A1 were inducible by 20 to 48 h treatment with β-naphthoflavone
(32µM), dexamethasone (8µM), and clofibrate (500µM),
respectively. Rat CYP 1A1, 1A2 and 3A1 were the most inducible forms (>100 fold). Human CYP 1A1 and 2Cs were
induced 3 fold by 48 h treatment with phenobarbital
(430µM). These results indicate that the rat and human cytochrome P450s are differentially inducible in this cell line
and that it should then represent a useful tool for evaluating
metabolism-toxicity relationships. We are currently investigating the protein profile of cytochrome P450 induction.
REFERENCES
1. Bravo, P., Bender, V. and Cassio, D.: Hepatology, 27,
576 (1998).
2. Decaens, C., Rodriguez, P., Bouchaud, C. and Cassio,
D.: J Cell Sci, 109, 1623 (1996).
3. Borde, F., Lagelle, F., Bender, V.E., Bouskila, M., Bonnet, M-C., Brizard, P., Masson, M-T., Chevalier, S.,
Charuel, C., Cassio, D., Biagini, C.P.: 11th North American ISSX meeting, Orlando, 27-31 October 2002,
Drug Metabol Rev, 34, supp. 1, 100 (2002).
MP58 THE DECREASE OF RAT LIVER
MICROSOMAL CYP3A2
CATALYTIC ACTIVITY DURING
AGING MAY BE THE
CONSEQUENCE OF AN
ENHANCED PROTEIN
INSTABILITY
gressively decreased until 24 months. At 24 months, the
CYP3A2 protein was decreased but levels of mRNA remained unchanged. The levels of carbonyl protein were increased in microsomes, while a decrease was observed in cytosolic proteins. Neither catalase nor the proteasome activity changed over the whole period, but GSH peroxidase
activity decreased. Taken together, these results suggest
that loss in enzyme activity may be explained by a decreased amount of CYP3A2 protein. Since levels of mRNA are
not modified during aging, the loss in proteins may be due
to either a decreased protein translation or an enhanced degradation. The former possibility is unlikely because protein synthesis rates (incorporation of radiolabeled leucine
into proteins) remained unchanged. We hypothesise that
CYP3A2 is modified during aging (protein carbonylation ?)
thus rendering the protein highly instable and subject to degradation by the proteasome. Indeed, we have recently reported that CYP3A2 is highly sensitive to changes in cellular environment3.
REFERENCES
1. Guenguerich FP, Ann. Rev. Pharmacol. Toxicol. 39, 1
(1999).
2. Cooper CO, Reil LM, Jayyosi Z, Bandiera S, Kelley M,
Ryan DE, Daniel R, McCluskey SA, Levin W, Thomas
PEArch. Biochem. Biophys. 301, 345 (1993) .
3. Rekka E, Evdokimova E, Eeckhoudt S, Labar G, Buc
Calderon P Biochem. Pharmacol. 64, 633 (2002).
VALÉRIE WAUTHIER, ROGER K. VERBEECK, and
PEDRO BUC CALDERON
Unité de Pharmacocinétique, Métabolisme, Nutrition et
Toxicologie (PMNT), Département des sciences pharmaceutique, Université Catholique de Louvain, 1200 Bruxelles,
Belgium ([email protected]).
CYP3A is one of the most important P450 enzymes that
metabolises approximately half the drugs in use today1.
Since CYP3A2 is the predominant expressed form in adult
male rats2, the aim of this work was to study the microsomal catalytic activity of CYP3A2 in rats of different ages
and to compare these results to levels of protein oxidation,
proteasome activity and antioxidant enzyme activities.
Microsomes and cytosol were prepared from livers of male
Wistar rats (HAN) with ages ranging from 3 to 24 months.
The activity of CYP3A2 was assayed by monitoring the
formation of hydroxylated metabolites of midazolam by
HPLC. The amount of CYP3A2 apoprotein was estimated
by Western Blot and the mRNA levels by RT-PCR. The
proteasome activity (chymotrypsin-like) was measured by
following the release of the fluorogenic AMC group after
proteolytic cleavage (excitation: 365 nm; emission: 450
nm). The protein carbonyl content was determined by immunoblotting after derivation with DNPH (diphenylhydrazine). Catalase was measured with the TiSO4 method and
GSH peroxidase by recording the NADPH oxidation. Midazolam hydroxylation increased from 3 to 9 months, remained constant between 9 and 18 months and then pro-
S107
MP59 COCAINE, BUT NOT
METAMPHETAMINE CAUSES
A CONCOMITTANT DECREASE
OF CYP2C AND CONSTITUTIVE
ANDROSTANE RECEPTOR
(CAR) EXPRESSIONS IN HUMAN
ASTROCYTOMA CELL LINE
U373 MG
C. MALAPLATE-ARMANDa,b, L. FERRARIa,
C. MASSONa, G. SIESTa, H. LAMBERTb, S. VISVIKISa,
and A.M. BATTa
a
Centre du Médicament, Inserm U525, Faculté de Pharmacie, Université Henri Poincaré Nancy I, 30 rue Lionnois,
54000 Nancy, France, bCentre d’Evaluation et d’Information sur la Pharmacodépendance, Hôpital Central, CO
N°34, 540035 Nancy cedex, France
Cocaine and metamphetamine are psychostimulant
abused drugs associated with a strong risk for intracranial
ischemic and hemorrhage stroke. Several unclear mechanisms may be responsible for this cerebrovascular complication including vasoconstrictive processes followed by
blood flow alterations1. Cytochromes P450, especially astroglial CYP2C may play role in modulating cerebrovascular functions, participating to vasodilatators metabolites
generation from arachidonic acid2. Mechanisms of
CYP2C mRNA regulation implicates two specific receptors: the constitutive androstane receptor (CAR) thought to
be inlvoved in phenobarbital induction and pregnane X receptor (PXR) thought to be involved in rifampicin induction3,4,5. In this work, we examined CYP2C regulation in
response to cocaine and metamphetamine in an astroglial
cell line model, by RT-PCR and western-blotting analysis.
U373 MG, a human astrocytoma cell line, expressed at
least two isoforms of CYP2C family namely CYP2C8
and CYP2C9. A 48 hrs treatment with cocaine (1, 10 and
100 µM) caused a significative total CYP2C, CYP2C8
and CYP2C9 mRNA decrease and a CYP2C9 protein level
decrease, whereas metamphetamine had no effect on
CYP2C mRNA expression. These effects of cocaine were
accompanied by an obvious repression of CAR mRNA
reaching - 90 % with 10 µM cocaine. Such data suggest
that this receptor is involved in CYP2C repression by cocaine, in this astroglial cell line. These findings represent
one possible molecular mechanism involved in cocaine
cerebrovascular effects and neurotoxicity.
REFERENCES
1. Petitti D.B., Sidney S., Quesenberry C., Bernstein A.:
Epidemiology. 9, 596, (1998).
2. Harder D.R., Zhang C., Gebremedhin D.: News Physiol. Sci. 17, 27, (2002).
3. Ferguson S.S., LeCluyse E.L., Negishi M., Goldstein
J.A.: Mol. Pharmacol. 62, 737, (2002).
4. Gerbal-Chaloin S., Pascussi J.M., Pichard-Garcia L.,
Daujat M., Waechter F., Fabre J.M., Carrere N., Maurel
P.: Drug Metab. Dispos. 29, 242, (2001)
5. Beigneux A.P., Moser A.H., Shigenaga J.K., Grunfeld
C., Feingold K.R.: Biochem. Biophys. Res. Commun.
293, 145, (2002).
MP60 FUNCTIONAL CHARACTERIZATION OF A SPLICED CYP3A7
VARIANT WITH A LONGER
C-TERMINAL, EXPRESSED IN
HUMAN FETAL LIVER
CRISTINA RODRÍGUEZ-ANTONAa,
MATS HIDESTRANDa, MAGNUS AXELSONb,
ANDERS RANEc and
MAGNUS INGELMAN-SUNDBERGa
a
Division of Molecular Toxicology, Institute of Enviromental Medicine, Karolinska Institute, SE-17177-Stockholm,
Sweden, bDivision of Clinical Pharmacology, Department
of Medicine, Karolinska Hospital, and cDivision of Clinical
Pharmacology, Huddinge University Hospital, SE-141 86
Stockholm, Sweden.
The human cytochrome P4503A (CYP3A) locus contains
three pseudogenes, CYP3AP1, CYP3AP2 and CYP3AP3 and
four genes CYP3A4, CYP3A5, CYP3A7 and CYP3A43. The
different CYP3A enzymes are expressed in a developmental and tissue-specific manner. In the fetal liver, CYP3A7
is the most abundant CYP, playing an important role in the
metabolism of steroids, in particular dehydroepiandrosterone (DHEA), and drugs. Recently, a CYP3A7 mRNA variant, consisting of the 13 exons of CYP3A7 and two additional exons at the 3’end from the CYP3AP1, was detected
in human liver1. This transcript would encode a CYP3A7
protein where the last 4 amino acids in the C-terminal have
been replaced by a unique sequence of 36 amino acids from
the two exons of the pseudogene. We found this variant incidentally when screening fetal liver and further examined
the expression using semi-quantitative RT-PCR. The
mRNA expression of the CYP3A7-variant was higher in
fetal livers than in adult livers and was not detected in human hepatoma cell lines. There was a high interindividual
difference in fetal hepatic expression and the variant overall represented 1% of CYP3A7 mRNA in the fetal livers.
The CYP3A7 and CYP3A7-variant proteins were heterologously expressed in yeast and in the mammalian HEK293
cells, and the activities of both enzymes were assayed
using [3H]DHEA. Unexpectedly the CYP3A7-variant was
catalytically very active and hydroxylated DHEA in the
7α-position in contrast to CYP3A7 which mainly formed
the 16α-OH product. Similar results were obtained both in
yeast and in HEK293 microsomes. It is concluded that
a trans-spliced product of the CYP3A7 gene is expressed in
fetal liver and that the corresponding enzyme exhibits
a different specificity as compared to CYP3A7.
REFERENCES
1. Finta C, Zaphiropoulos PG: Gene 260, 13, (2000).
S108
suggesting a possible peroxisomal proliferator response to
components of RPI. Supported in part by ARS CRIS
#6251-51000-003-065 and Riceland Foods, Inc.
MP61 EFFECTS OF WEANING TO
DIETS CONTAINING RICE
PROTEIN ISOLATE (RPI) ON
GROWTH, PLASMA IGF1 AND
EXPRESSION OF CYP2C11 AND
CYP4A1 IN RAT LIVER
REFERENCES
MARTIN J.J. RONISa,b, MISTY REEVESa,
HOLLY HARDYa, JAMIE BADEAUXa, CHRIS DAHLa,
DAVID HARISSONa, RANI HALEYa,
LANDON HUMPHREYa, MAT FERGUSONa, and THOMAS M. BADGERa,c
a
Arkansas Children’s Nutrition Center, bDepartment of
Pharmacology and Toxicology, cDepartment of Physiology,
University of Arkansas for Medical Sciences, Little Rock,
AR, USA., [email protected]
Rice contains a number of phytochemicals including
vitamin E derivatives, tocotrienols and oryzanols which
have been suggested to contribute to health beneficial effects of rice consumption including reductions in cholesterol1,2. In addition, protein isolates from rice are in widespread use in the food industry; have been shown to produce
dietary protection against DMBA-induced mammary tumorigenesis in rats3,4; and have many associated phytochemical constituents5. We fed groups of time-impregnated female Sprague-Dawley rats pelleted AIN93G diets with casein as the sole protein source ad libitum from gestational
d. 4 until their pups weaned at post-natal d.21. Litters were
culled to 5 male and 5 female pups and N = 8-10 male or
female pups were weaned onto amino acid matched pelleted, diets, containing either casein (CAS) or rice protein
isolate (RPI) as the sole protein source, ad libitum beginning on post-natal d. 15 until sacrifice at post-natal d. 34.
Body weight gain was measured and serum IGF1 concentrations were assessed by radioimmunoassay. In addition liver microsomes were prepared. Testosterone 16α-hydroxylase was measured in male pups as an indicator of
CYP2C11-dependent monooxygenase activity, CYP2C11
apoprotein was immunoquantified by Western blot and
CYP2C11 mRNA was quantitated by real time RT-PCR. In
addition, CYP4A1 apoprotein and mRNA were measured
in liver microsomes from male and female pups by Western
blot and real time PCR. Feeding RPI reduced body weight
gain in both male and female pups (p < 0.05). This was
accompanied by reductions in plasma IGF1 concentration
(p < 0.05) indicating a suppression or delay in development
of the GH/IGF1 axis in these animals. CYP2C11 mRNA
and apoprotein expression and testosterone 16α-hydroxylase were also inhibited in male pups (p < 0.05). Since food
intake was not measured it is unclear if this effect was due
to reduced intake, reduced dietary energy availability or
a direct effect of RPI on GH secretion. CYP4A1 mRNA
and apoprotein were increased 4-5-fold in pups of both sex
S109
1. Qureshi A.A., Peterson D.M., Hasler-Rapacz J.O., Rapacz J.: J. Nutr. 131, 223 (2001).
2. Xu Z., Hua N., Godber J.S.: J. Agric. Food Chem. 49,
2077 (2001).
3. Morita T., Kiriyama S.: J. Nutr. Sci. Vitaminol. 42, 325
(1996).
4. Hakkak R., Korourian S., Fletcher T., Hale K, et al.:
AACR Proc. 43, 823 (2002).
5. Fang N., Yu S., Nehus Z., Badger T.M.: FASEB J. 16,
A1011 (2002).
MP62 EFFECTS OF WEANING TO
DIETS CONTAINING SOY
PROTEIN ISOLATE (SPI) OR
ISOFLAVONES ON GROWTH,
PLASMA IGF1 AND
EXPRESSION OF CYP2C11
AND CYP4A1 IN RAT LIVER
MARTIN J.J. RONISa,b, MISTY REEVESa,
HOLLY HARDYa, JAMIE BADEAUXa, CHRIS DAHLa,
DAVID HARISSONa, RANI HALEYa,
LANDON HUMPHREYa, MAT FERGUSONa, and
THOMAS M. BADGERa,c
a
Arkansas Children’s Nutrition Center, bDepartment of
Pharmacology and Toxicology, cDepartment of Physiology,
University of Arkansas for Medical Sciences, Little Rock,
AR, USA., [email protected]
Soy protein isolate (SPI+) is fed to over 1 million children each year in the U.S. as part of soy-infant formula. Soy
has been shown to have a number of health beneficial effects including reductions in cholesterol1 and has been suggested to increase lean body mass2,3. The health effects have
appear to be associated with both the soy protein component itself and to isoflavone phytoestrogens bound to SPI + 1.
In the current study we fed groups of time-impregnated female Sprague-Dawley rats pelleted AIN93G diets with casein as the sole protein source ad libitum from gestational
d. 4 until their pups weaned at post-natal d.21. Litters were
culled to 5 male and 5 female pups and N = 8-10 male or
female pups were weaned onto amino acid matched, pelleted, diets containing either casein (CAS) or SPI+, ad libitum beginning on post-natal d. 15 until sacrifice at post-natal d. 34. Other groups were fed SPI stripped of phytochemicals by ethanol washes (SPI-) or CAS diets supplemented with the soy-isoflavones genistein or daidzein at a level
of 1 mg/kg Body weight gain and serum IGF1 concentrations were measured. CYP2C11-dependent testosterone 16_hydroxylase was measured in liver microsomes from male
pups, CYP2C11 apoprotein was quantified by Western blot
and CYP2C11 mRNA by real time RT-PCR. In addition,
CYP4A1 apoprotein and mRNA were measured in male
and female pups and microsomal CYP4A1-dependent lauric acid 12-hydroxylase was assessed. Feeding SPI+ or SPIreduced body weight gain in both male and female pups
(p < 0.05). This was accompanied by reductions in plasma
IGF1 indicating a suppression or delay in development of
the GH/IGF1 axis in these animals. CYP2C11 mRNA and
apoprotein expression and testosterone 16α-hydroxylase
were also inhibited in male pups fed SPI+ or SPI- (p < 0.05).
Since food intake was not measured it is unclear if this effect was due to reduced intake, reduced dietary energy availability or a direct effect of SPI on GH secretion. No such
effects were observed in pups fed CAS + gesistein or daidzein. Lauric acid 12-hydroxylase; CYP4A1 apoprotein and
mRNA expression were inhibited (p < 0.05) in pups of both
sex fed SPI+ but not SPI- or isoflavones suggesting a possible inhibition of peroxisomal proliferation response by
non-isoflavone phytochemical components bound to SPI.
Supported in Part by ARS CRIS #6251-51000-003-065 and
DuPont Protein Technologies.
REFERENCES
1. Clarkson TB: J. Nutr. 132, 566S (2002).
2. Bhathena SJ, Velasquez MT: Am. J. Clin. Nutr. 76, 1191
(2002).
3. Payne RL, Bidner TD, Southern LL, Geaghan JP: J.
Animal Sci. 79, 1230 (2001).
MP63 HUMAN CRYOPRESERVED
HEPATOCYTES AS AN IN VITRO
MODEL TO STUDY
CYTOCHROME P450
INDUCTION
induction in preclinical development, their supply is often
erratic and/or their condition is in many cases poor. In addition, assay standardization is rendered more difficult due
to a high interindividual variation of CYP expression in hepatocytes. Improvement of cryopreservation methods and
optimalization of culturing conditions have led to the possibility to culture viable and plateable hepatocytes after
cryopreservation. A major advantage for the pharmaceutical industry is that long-term induction experiments can be
planned precisely and that the hepatocyte populations used
in these studies can be standardized easily. Human cryopreserved hepatocytes (HCH’s) from four different donors
were plated, the cells were allowed to recover for 48h and
then incubated with 50 µM omeprazole, 25 µM rifampicin,
10 µM dexamethasone, 100 µM phenobarbital, 100 µM clofibric acid or 150 mM ethanol for another 48h and compared to non-treated cells. CYP1A2, CYP2B6, CYP2C9,
CYP2C19, CYP2E1 and CYP3A4 induction was determined by measuring 7-ethoxyresorufin-O-demethylation,
[14C ]s-mephenytoin-N-demethylation, [14C]tolbutamidemethyl-hydroxylation, [14C ]s-mephenytoin-4’-hydroxylation, chlorzoxazone-6-hydroxylation and [14C]testosterone6(-hydroxylation respectively. In addition, mRNA and protein expression was analysed by TaqMan QRT-PCR and
immunodetection. In general, HCH’s responded well to
the respective typical inucers. For example, activity, protein and mRNA expression of batch 1 was increased 13-,
25-, and 79-fold respectively upon omeprazole treatment.
A 2- to 3-fold induction was observed of CYP2C9 mRNA
after rifampicin and phenobarbital treatment, while maximally a 17-, 6-, and 24-fold induction was measured of
CYP3A4 upon treatment with the same typical inducers.
Induction profiles of the different HCH-batches will be discussed in detail. In conclusion, HCH’s may be a good
alternative to fresh hepatocytes to study CYP induction.
MP64 CYP1A REGULATION BY
α-TOCOPHEROL
YULIA A. SIDOROVA, and ALEVTINA Y. GRISHANOVA
Institute of Molecular Biology and Biophysics, Siberian
Division of Russian Academy of Medical Sciences, Novosibirsk, Russia, Timakova str., 2, [email protected]
DIRK ROYMANS, PIETER ANNAERT,
JOS VAN HOUDT, ADRI WEYGERS,
DIETSKE JACOBS, JAN HENDRICKX,
GEERT MANNENS, and WILLEM MEULDERMANS
Preclinical Pharmacokinetics, Johnson & Johnson Pharmaceutical Research & Development, a division of Janssen
Pharmaceutica N.V., Turnhoutsebaan 30, B-2340 Beerse,
Belgium, [email protected].
Major restrictions are connected to current in vitro models for cytochrome P450 (CYP) induction screening during drug development. Although fresh human hepatocytes
are still considered as ‘the golden standard’ to study CYP-
S110
CYP1A play dominant role in the bioactivation of polycyclic aromatic hydrocarbons (PAH) and heterocyclic amines, widespread chemical carcinogens, to genotoxic derivatives. Until the recent times only planar aromatic chemicals
considered among CYP1A1 inducers. At the last 2 decades
was shown that compounds with other structure might induce CYP1A1. CYP1A1 induction by PAH is mediated by
Ah receptor, transcription factor regulated CYP1A1 gene
expression. In the present study effects of α-tocopherol on
rat hepatic CYP1A were investigated on male Wistar rats
weighting 100 - 120 g. Oil solution of α-tocopherol was administered per os in dose 150 mg/kg body weight daily,
once or during 12 days. CYP1A1-dependent 7-ethoxyresorufin O-deethylase (EROD), CYP1A2-dependent 7-methoxyresorufin O-demethylase (MROD) and NADPH-cytochrome P450 reductase activities were measured in rat hepatic microsomes. CYP1A1 and CYP1A2 mRNA levels in
the liver of control and experimental animals were measured by semi-quantitative RT-PCR technique. Formation of
Ah receptor complex with oligonucleotide XRE3, containing 3 repeats of DNA sequence, which specifically binds
with AhR, in the liver nuclear extracts was determined
by gel-retardation technique. The single α-tocopherol administration led to 3,5-fold enhancement of EROD activity;
3,3-fold of MROD activity and had no effect on NADPHcytochrome P450 reductase activity. 12 days long administration of α-tocopherol led to 2-fold enchancement of
EROD activity and had no effect on MROD and NADPHcytochrome P450 reductase activities. CYP1A1 mRNA level in the liver of experimental animals was significantly
higher than in control after 12 days long administration of
α-tocopherol, though this treatment has no effect on
CYP1A2 mRNA level. Ah receptor complex with XRE3
was detected only in rat liver nuclear extracts after single
α-tocopherol administration. No complex formation was
observed in control nuclear extracts or experimental after
12 days long administration of α-tocopherol. Thus, enhancement of CYP1A1 activity and mRNA level under α-tocopherol action is mediated by Ah receptor.
Supported by grant RFBR N 02-04-48639.
MP65 THE ROLE OF CYTOCHROMES
P450 IN BIOTRANSFORMATION
OF NEW ANTILEUKOTRIENIC
DRUG QUINLUKAST IN RAT
Fig. 1,
transformation study of Q in male rats in vitro had revealed that Q sulfoxide is the main metabolite identified in
microsomal fraction and in isolated hepatocytes. Also other metabolites of Q were found, which structure is still not
known (M3, M5). The present study was conducted to characterise the enzymes involved in Q biotransformation in
isolated rat hepatocytes.
The primary cultures of rat hepatocytes were 48 hours
treated by inducers of different CYPs (rifampicin, dexamethasone, phenobarbital, ethanol, β-naphthoflavone).
Then Q (50 µM) was incubated 24 hours in primary culture
of induced or control hepatocytes. The effects of CYPs inhibitors ketoconazole, methylpyrazole, metyrapone and
α-naphthoflavone (2, 10, 50 µM) on Q metabolism were
tested in induced and control hepatocytes.
Significant induction of Q S-oxide formation (6 times)
by dexamethasone and strong concentration-dependent inhibition by ketoconazole was observed. Dexamethasone
also induced formation of metabolite M5 (twice) and ketoconazole strongly inhibited this metabolite. CYP1A inducer, β-naphthoflavone, induced 10 times production of metabolite M3 and strong inhibition effect of α-naphthoflavone on its formation was observed. These results indicate
that CYP3A catalyse sulfoxidation of Q and formation of
its metabolite M5 and CYP1A catalyse formation of metabolite M3 in primary culture of rat hepatocytes.
This project was supported by Czech Ministry of Education, Research Centre LN00B125.
REFERENCES
BARBORA SZOTÁKOVÁ, LENKA SKÁLOVÁ,
LUDùK ·I·PERA, and VLADIMÍR WSÓL
Department of Biochemical Sciences, Research Centre
LN00B125, Charles University, Faculty of Pharmacy,
Heyrovského 1203, CZ-500 05 Hradec Králové, Czech
Republic, [email protected]
Anti-leukotrienes drugs have become available for the
clinical management of asthma. They are functioning either by blocking the interaction of leukotrienes with receptors or by inhibiting leukotriene synthesis. The new promising drug belonging to anti-leukotrienes is quinlukast 1 (Q),
4-(4-(quinoline-2’-yl-methoxy)phenylsulphanyl)benzoic
acid (VÚFB 19363), Fig. 1. It is now under investigation
for its anti-inflammatory and anti-asthmatic effects. In comparison to the specific effect of montelukast and zafirlukast,
Q displays multiple antileukotrienic effects2. Phase I bio-
S111
1. Kuchar M., Culikova K., Panajotova V., Brunova B.,
Jandera A., Kmonicek.: Coll. Czech. Chem. Commun.
63, 103 (1998).
2. Kuchar et al, manuscript in preparation
drug was detected only in abomasal fluid. The repeated administration of ABZ to mouflons did not affected ABZ sulfoxidation in microsomes but oxidation of ABZSO was
significantly faster in microsomes from ABZ treated animals.
We can conclude that administration of repeated doses
of ABZ led to acceleration of its own metabolism probably
due to CYP1A induction. Increasing deactivation of ABZ
could result in the failure of therapy.
This work was supported by Grant Agency of the
Czech Republic, Grant No. 524/03/1361 and Grant Agency
of Charles University, Grant No.96/2002/C/FAF.
MP66 EFFECT OF REPEATED
ALBENDAZOLE ADMINISTRATION TO MOUFLONS ON
ALBENDAZOLE METABOLISM
IN VIVO AND IN VITRO
JAKUB VELÍKa, LENKA SKÁLOVÁb,
VENDULA BALIHAROVÁb, BARBORA
SZOTÁKOVÁb, VLADIMÍR WSÓLb, and JI¤Í LAMKAa
a
Department of Pharmacology and Toxicology, bDepartment of Biochemical Science, Faculty of Pharmacy, Charles University, Heyrovského 1203, Hradec Králové, Czech
Republic, [email protected]
Albendazole (ABZ) is a benzimidazole derivate with
a broad-spectrum activity against animal helminth parasite.
The effective treatment of polygastric animals can be often
reached by a single dose administration of ABZ but treatment
of some resistant parasitic diseases (e.g. small lungworms
and small liver flukes) require repeated drug administration.
After oral administration in animals, ABZ is metabolised to
biologically active albendazole sulfoxide (ABZSO) in two
enantiomeric forms: (–)-ABZSO and (+)-ABZSO. It is
known that the CYPs (mainly CYP3A) system generates
(–)-ABZSO, while the FMO system selectively produces
(+)-ABZSO. CYP1A mediate ABZSO oxidation to inactive albendazole sulfone (ABZSO2)1. Inter-species differences in quantitative aspects as well as stereospecificity of
ABZ metabolism was described2,3. In laboratory animals
and human cells ABZ administrations caused induction of
CYPs, mainly CYP1A4,5.
The aim of this project was to study effect of repeated
ABZ administration on its oxidative metabolism.
Adult mouflon ewes (8 animals) from game enclosure
Vlkov (Czech Republic) were divided into two groups:
first group was treated repeatedly by therapeutic doses of
ABZ (p.o. 5 ( 7.5 mg/kg of body weigh), second group represented control. Repeatedly, 24 h after each dose, blood
samples were collected and metabolites were quantified by
HPLC. Animals were culled 24 h after the last dose. The
bile from gall bladder, blood, and ruminal fluid were sampled for HPLC analyses. Whole liver and small intestine
were removed immediately and stored in liquid nitrogen
during transport to laboratory. Microsomes were prepared
from homogenate of both tissues of individual animals. In
vitro biotransformation of ABZ and ABZSO were tested in
liver and intestinal microsomes.
Plasma level of total ABZSO did not significantly
change during whole experiment (5 days), but the relative
proportion of (+)-ABZSO enantiomer significantly increased. Increasing ABZSO2 plasma concentration in dependence on number of ABZ doses were also observed. In bile,
ABZSO2 and both ABZSO enantiomers were found in concentration similar to their concentration in plasma. Parent
S112
REFERENCES
1. el Amri H.S., Fargetton X., Delatour P., Batt A.M.: Xenobiotica 10, 1159 (1987).
2. Delatour P., Benoit E., Besse S., Boukraa A.: Xenobiotica 2, 217 (1991).
3. Delatour P., Garnier F., Benoit E., Caude I.: Res. Vet.
Sci. 2, 134 (1991).
4. Souhaili el Amri H., Mothe O., Totis M., Masson C.,
Batt A.M., Delatour P., Siest G.J.: Pharmacol. Exp.
Therap. 2, 758 (1988).
5. Asteinza J., Camaco-Carrnza R., Reyes-Reyes R.E.,
Dorado-González V., Espinosa-Aguirre J.J.: Environ.
Toxicol. Pharmacol. 9, 31 (2000).
MP67 AGE RELATED CHANGES IN
THE PROTEIN AND MRNA
LEVELS OF CYP2E1, CYP3A1,
CYP3A2 AS WELL AS IN THEIR
HEPATIC ACTIVITIES IN
WISTAR RATS
VALÉRIE WAUTHIER, NICOLE MATON,
VÉRONIQUE ALLAEYS, ROGER K. VERBEECK, and
PEDRO BUC CALDERON.
Unité pharmacocinétique, métabolisme, nutrition, toxicologie (PMNT), Département des sciences pharmaceutiques, Université catholique de Louvain, Belgium
[email protected]
The biotransformation of a drug and, therefore its therapeutic effect, may be modified during ageing. Among
different causative factors, the impairment of normal cellular functions by free radicals has been evoked as playing
a critical role. The aim of our study was to evaluate the effect of age on the expression and activity of CYP2E1,
CYP3A1 and CYP3A2. Total cytochrome P450, protein
and mRNA levels and activities for each isoform were investigated. In addition, we determined some markers of
oxidative stress like TBARS (thiobarbituric acid reactive
substances), histochemical detection of aldehydes, carbonyl proteins and GSH content. Liver microsomes were prepared from male Wistar rats of 3, 8, 11 and 18 months old.
Chlorzoxazone was used as a probe to assess CYP2E1
activity and midazolam for CYP3A1 and CYP3A2 activities. Metabolite formation, 6-hydroxy-chlorzoxazone for
chlorzoxazone and α-OH midazolam and 4-OH midazolam
for midazolam was quantified by HPLC. Protein isoform
contents were determined by Western Blot and mRNA levels by RT-PCR. Lipid peroxidation was assessed following
the formation of TBARS and by histochemical detection
of aldehydes, the direct Schiff’s reaction. Protein carbonyl
content was determined by measuring at 355 nm the
formation of hydrazone after reaction with DNPH (diphenylhydrazine). GSH level was estimated fluorimetrically by
determination of the thiol content by reaction with OPT
(o-phtaldialdehyde). Results revealed that total cytochrome
P450, the expression and the activity of CYP3A1 and
CYP3A2 did not change until 18 months. Nevertheless,
chlorzoxazone hydroxylation increased from 3 to 8 months,
remained constant between 8 and 11 months and then progressively decreased until 18 months. Interestingly, microsomal protein and mRNA level of CYP2E1 did not change
over the whole period. While the amount of proteins did not
change, their functionality may be affected by oxidative
stress (increase in TBARS and histochemical aldehyde detection, decrease in GSH level). However, we did not observe any change in carbonyl protein content. The decrease
in chlorzoxazone biotransformation in rats after 11 months
is most probably due to post-translational modifications of
CYP2E1 proteins and well correlated with the accumulation
of damage due to oxidative stress. Since no change in the
midazolam α- and 4- hydroxylation activities as well as protein and mRNA content was observed for the CYP3A1 and
CYP3A2 until 18 months, it seems that such isoforms are
less affected by the oxidative environment.
MP68 ADMINISTRATION OF CYP19
MODULATORS TO
GALLUS DOMESTICUS
TOMÁ· KOBLASa, PAVEL TREFILb,
MARIE STIBOROVÁa, and PETR HODEKa
a
University, Albertov 2030, 128 40 Prague 2, The Czech Republic, bBIOPHARM, Research Institute of Biopharmacy
and Veterinary Drugs, a. s., Pohofií ChotouÀ, CZ-254 49
Jílové, The Czech Republic, [email protected],
[email protected]
Estrone, one of female sex hormones, is formed by
a unique cytochrome P450 CYP19 which builds a phenolic
ring A of all estrogens. This enzyme is present in ovarian
tissue of birds. The modulation of CYP19 activity might
S113
significantly change the estrone/testosterne ratio and consequently is expected to change the poultry sex phenotype.
For example, in ovo administration of CYP19 inhibitor,
Fadrazole, elicited testes formation in genotype females.
Female masculinisation resulting in lowered body fat and
increased volume of muscles might be important in the respect of the chicken meat production. Natural plant compounds, flavonoids, were selected as potential CYP19 inhibitors. Their inhibitory capacity towards CYP activities has
been extensively studied because of their potential use as
agents blocking the initiation stage of carcinogenesis.
Synthetic and naturally occurring flavonoids are effective
inhibitors of mammalian CYP1A1, 1A2, 1B1 and 3A4 as
well as CYP19. The presence of C4 oxo-group of the flavonoid skeleton, which is approaching the heme iron (based on models), seems to be crucial for inhibition. One can
expect that the substitution of oxygen for sulphur in this
position will result in a higher binding capability. Hence,
synthetic flavonoids, 7,8-benzoflavone (ANF) and its thioanalogue (SANF), were tested in vivo as food additive for
hatched animals. Resulting changes of CYP expression,
steroid hormone levels, body composition, and chicken behavior were monitored. Short-time i.p. administration of
ANF, SANF and 5,6-benzoflavone (BNF), used for comparison, caused large induction of chicken CYP crossreacting
with antibodies against rat CYP1A1. CYP recognized by
anti-rabbit CYP2B4 antibody was elevated, too, while
CYPs detected by antibodies against rabbit CYP3A6 and
2E1 were not significantly affected. These results were
confirmed with metabolic data: 7-ethoxyresorufin deethylase activity was increased 10-30 times by application of
flavonoids, showing BNF to be most efficient one. Effect
of long-time p.o. treatment of animals with ANF and
SANF in respect to CYP expression in various organs is
being currently examined. In case of female chicken both,
ANF and SANF, caused induction of CYP1A4, while
SANF induced CYP1A5 in male chicken. There were no
significant changes detected by antibodies against rabbit
CYP3A6 and 2E1.
The work was supported by the grants 523/01/0840
from The Grant Agency of The Czech Republic, and
MSM-113100001 from The Czech Ministry of Education.
04 PostTP.QXD 10.6.2003 12:01 Stránka 114
Poster Presentations – TP
6
POSTERS – TUESDAY – TP
TP01
NON-DISSOCIATIVE
SEQUENTIAL METABOLISM
OF AN 8-ALKYL XANTHINE BY
HUMAN P450 1A21
determined. The analytic solution of the scheme using the
steady state assumption was transformed using the Cleland
nomenclature into a form that accepted the measured apparent Michaelis-Menton rate constants. The results of this
effort were estimates of the rate constants for all binding
and catalytic steps. Branching ratios determined from our
estimates of the true internal rate constants compared favorably with the branching ratios from the isotope dilution
experiments. These results suggest a role for CYP1A2
active site water in the conversion of olefin to diol via the
putative epoxide intermediate. A similar role for active site
water in the deactivation of reactive imidazo-alkide intermediates produced by the metabolism of other 8-alkylxanthines such as furafylline by CYP1A2 has been proposed4.
JASON BOER, KYLE E. ALLEN, and
KENT L. KUNZE
Department of Medicinal Chemistry, Box 357610,
University of Washington, Seattle, Washington USA
[email protected]
REFERENCES
Cytochrome P450 enzymes are versatile catalysts with
broad substrate specificities. It is not unusual to find that
a product of a P450-catalyzed reaction may undergo further metabolism by P450 enzymes. Examples where the sequence of oxidation reactions is catalyzed by the same enzyme and where the initial substrate undergoes multiple
oxidation events before dissociation from the enzyme are
found among the steroid metabolizing P450. For instance,
P450arom and P450scc are known to catalyze three successive
oxidation reactions on the same substrate molecule prior to
releasing the final product. Examples of non-dissociative
sequential metabolism amongst the xenobiotic metabolizing P450 enzymes are relatively rare. An interesting
example is found in the conversion of ethanol to acetic acid
by CYP2E12.
We have recently discovered a unique example of sequential metabolism where CYP1A2 catalyzes a sequence
of three oxidative reactions directed towards the isopropyl
sidechain of an 8-alkyl xanthine (IPCH) (Figure 1). Isotope
dilution methodologies3 were employed in order to deter-
1. Supported by NIH Grants GM32165 and GM48750
2. Bell-Parikh, L.C., Guengerich F.P.: J. Biol. Chem. 34,
23833 (1999).
3. Sugiyama, K., Nagata, K., Gillette J.R., Darbyshire J.F.:
Drug Metab. Dispos. 22, 584 (1994).
4. Racha, J.K., Kunze, K.L.: J. Amer. Chem. Soc. 21, 5337
(1998).
TP02
CAFFEINE OXIDATION DURING
PROLONGED EXPOSURE OF
RATS TO PHENOTHIAZINE
NEUROLEPTICS
W¸ADYS¸AWA ANNA DANIEL, MARTA KOT, and
JACEK WÓJCIKOWSKI
Polish Academy of Sciences, Institute of Pharmacology,
Department of Pharmacokinetics and Drug Metabolism,
[email protected]
Fig. 1.
mine the true branching ratios for each reaction in the sequence. This analysis revealed that substantial fractions of
the olefin (77%) and diol (32%) that are formed in the enzyme active site are sequentially converted to the next metabolite (diol and ketone respectively) before they can be
released from the enzyme. Overall, 25% of the first-formed
primary olefin metabolite is converted to the tertiary ketone metabolite within the confines of the enzyme active
site. Branching ratios (kcat/koff) were found to be highly sensitive to rates of electron transfer. Apparent Km and kcat values for substrates IPCH, olefin and diol were separately
S114
Because of its natural character, caffeine is used as
a test substance for estimation of metabolic phenotype with
regard to the activity of NAT2 and CYP1A2 in humans and
rats. The primary metabolic pathways of caffeine are 3-Ndemethylation to paraxanthine (CYP1A2), 1-N-demethylation to theobromine and 7-N-demethylation to theophylline
(CYP1A2 and other enzymes), and C-8-hydroxylation to
1,3,7-trimethyluric acid (mainly CYP3A). There are some
suggestions that 1-N-demethylation and 7-N-demethylation engage probably also isoenzymes CYP2B and/or
CYP2E1. Our previous studies indicated that phenothiazine neuroleptics given in vitro to rat liver microsomes inhibited the above-described oxidation pathways of caffeine
metabolism. The aim of our present work was to study the
effect of 24 h-exposure and chronic treatment with pharmacological doses of classic neuroleptics (phenothiazines
with different side chains and aromatic ring substituents)
on the activity of rat cytochrome P-450 measured by caffeine oxidation. The investigated neuroleptics were administered intraperitoneally (ip.), twice a day for one day or two
weeks at the following pharmacological doses (mg/kg i.p.):
promazine, levomepromazine, thioridazine and perazine 10,
chlorpromazine 3. The rats were sacrificed at 12 h (one-day
treatment) or 24 h (two-week treatment) after the drug
withdrawal, and liver microsomes were prepared by differential centrifugation in 20 mM Tris/KCl buffer (pH = 7.4),
including washing with 0.15 M KCl, according to a conventional method. The in vitro metabolic studies were carried out at linear dependence of the metabolite formation
on time, and protein and substrate concentrations. The rates of 3-N-, 1-N- and 7-N-demethylation and C-8-hydroxylation of caffeine were assessed at a substrate concentration
of 800 µM. Caffeine and its four oxidized metabolites were
assessed using the HPLC method (Daniel et al. 2001) adapted from Rasmussen and Brøsen (1996). One-day (i.e. 24 h)
exposure to phenothiazine neuroleptics resulted in a significant increase in the rates of the four oxidation pathways
of caffeine by promazine and perazine. Moreover, thioridazine significantly increased the rates of 1-N- and 3-N-demethylation, while chlorpromazine enhanced that of C-8hydroxylation of caffeine. Levomepromazine did not produce any significant changes in the rate of caffeine oxidation. After two-week treatment with the investigated phenothiazines, the increased rates of caffeine oxidation pathways, which appeared after one-day exposure, were still
maintained, with an exception of the thioridazine effect on
1-N- and 3-N-demethylation. The obtained results indicate
that, apart from a direct effect of phenothiazine neuroleptics on the activity of cytochrome P-450 (competitive or
mixed inhibition), prolonged exposure of rats to those
drugs may stimulate the activity of its different isoforms,
i.e. of CYP1A2, CYP3A, and probably of other ones. Thus,
the effect of the phenothiazine neuroleptics on the activity
of cytochrome P-450 in vivo will be a resultant of their direct inhibitory effect on the enzyme (dependent on a current drug concentration) and the induction level (time-dependent). Adequate studies are in progress to identify CYP
isoforms responsible for the catalysis of particular pathways of caffeine oxidation, which should allow for a full
interpretation of the changes in CYP activity observed after neuroleptic treatment. It seems also interesting to find
out whether similar effects develop during long neuroleptic therapy of schizophrenic patients.
TP03
ACTIVITY OF NADPH-CYTOCHROME P-450 REDUCTASE
IN THE HUMAN HEART, LIVER
AND LUNGS IN THE PRESENCE
OF PADMA 28 – IN VITRO
STUDY
JAROS¸AW DUDKAa, MAREK MURIASb,
FRANCISZEK BURDANc, JUSTYNA SZUMI¸¸Od,
ROBERT KLEPACZd , JADWIGA JODYNIS-LIEBERTb
a
Department of Toxicology, Medical University of Lublin,
PL-20093 Lublin, [email protected], bDepartment of Toxicology, University of Medical Sciences Poznaƒ, PL-60631 Poznaƒ, cExperimental Teratology Unit,
Medical University of Lublin, PL-20074, dDepartment of
Pathomorphology Medical University of Lublin, PL-20090
Lublin, Poland
Padma 28 is a mixture of herbs used in traditional Tibetan medicine with anti-inflammatory activity. It was showed previously1 that Padma 28 induced a concentration dependent inhibition of nitric oxide synthase (iNSO) protein
expression and iNSO mRNA levels. NADPH-cytochrome
P-450 reductase and iNOS have many structural similarities and consequently both can catalyze redox cycling and
bioreductive anticancer agent2. The aim of this study was
to investigate the activity of NADPH-cytochrome P-450
reductase in the presence of Padma 28. The NADPH-cytochrome P-450 reductase was obtained from human hearts,
lungs and livers post mortem. The activity of NADPHcytochrome P-450 reductase was evaluated by measuring the
rate of cytochrome c reduction at 550 nm at 37 oC (pH=7.4).
First, the activity of the enzymes alone was measured. Subsequently, activity of NADPH-cytochrome P-450 reductase
was examined in the presence of different volumes of
Padma 28 (1.0; 2.0; 5.0; 10.0 µl in total volume 1000 µl).
Our study showed that Padma 28 increased NADPH-cytochrome P-450 reductase activity in the human samples
with all tested doses. In all cases, the enzyme activity was
enhanced with the rise of given concentrations. Padma 28
elevated a catalytic efficiency in increasing order of
lung>liver>heart.
REFERENCES
1. Moeslinger T., Friedl R., Volf I., et all.: Can. J. Physiol.
Pharmacol. 86, 78 (2000).
2. Garner A.P., Paine M.J.I., Rodriguez-Crespo I. et all.:
Cancer Research 1929, 59 (1999).
S115
TP04
ETHANOL INFLUENCE ON
METHANOL OXIDATION VIA
ADH AND MEOS
we could try to explain these controversies, in theory. Ethanol can inhibit ADH activity but also it can activate
MEOS activity and methanol oxidation could change the
predominant pathway of biotransformation.
KATARZYNA DAWIDEK-PIETRYKAa,
JAROS¸AW DUDKAa, and KATARZYNA DOMA¡SKAb
TP05
a
PL-20-093 Lublin, bDepartment of Poultry Diseases, National Veterinary Institute, Pulawy
[email protected],
The liver is the major organ responsible for methanol oxidation, and alcohol dehydrogenase (ADH) and microsomal
alcohol oxidising system (MEOS) cytochrome P-450 - depended are the main enzymes involved. Methanol toxicity is
directly caused by its toxic metabolites and the major task in
clinical toxicology is to inhibit each of these enzymes to protect human life. Ethanol, well-known competitive inhibitor of
ADH administered in methanol intoxication has raised many
contradictory opinions. Generally it is not an ideal antidote.
Our study is a part of the project involved in searching for
new solutions in the methanol intoxication treatment based
on postulate that MEOS is also involved in methanol oxidation, in addition to ADH. In the present study human liver
samples were homogenised and used for enzymatic assays.
ADH activity was assayed by methanol monitoring, GC method. MAOS activity was measured by formaldehyde monitoring, spectrophotometrical method. The percentage of enzymes activity changes was calculated for ethanol at concentrations 0,05, 0,022 , 0,01, 0,005 and 0,001 M. In order to calculate kinetic parameters the data were plotted according to
Lineaweaver-Burk and Dixon. The activities of ADH and
MEOS were measured by reaction with methanol ranging
from 0,01 to 1,0 M or 0,03 to 0,5 M, respectively. Significant
differences of enzymes activities were observed. The mean
value of ADH activity was 1 µKat/g tissue and for MEOS activity 3 µKat/g tissue. The differences in Km and Vm for both
enzymes are significant. A higher ADH Km value (53,6 mM)
indicates that methanol has lower affinity to ADH as compared to MEOS (Km 6,7 mM), in vitro. However, Vm is much
higher for methanol oxidation catalysing by ADH. The results of the present study and data obtained from other authors allowed us to compare kinetic parameters for methanol
and ethanol as ADH and MEOS substrates. The present results confirm that ethanol compared to methanol has higher
affinity to ADH, by contrast to MEOS. As recent investigation, our study showed that ADH was competitively inhibited
(Ki 24,8 mM) to different extents by all examined concentrations of ethanol in reaction with 0,05M and 0,10 M methanol. On the contrary, ethanol is responsible for increasing
of MEOS activity with methanol at concentrations of
0,05 M and 0,20 M. There are a lot of doubt because the effectiveness of ethanol therapeutic administration in methanol intoxication. Specially, in alcoholics this problem
could be observed. However, in light of our investigation
S116
ACTIVATION OF NADPH-CYTOCHROME P-450 REDUCTASE
IN THE HUMAN HEART, LIVER
AND LUNGS BY UKRAIN – IN
VITRO STUDY
JAROS¸AW DUDKAa, MAREK MURIASb,
FRANCISZEK BURDANc, JUSTYNA SZUMI¸¸Od,
ROBERT KLEPACZd ,
and JADWIGA JODYNIS-LIEBERTb
a
PL-20093 Lublin, bDepartment of Toxicology, University of
Medical Sciences Poznaƒ, PL-60631 Poznaƒ, cExperimental Teratology Unit, Medical University of Lublin,
PL-20074, dDepartment of Pathomorphology Medical University of Lublin, PL-20090 Lublin, Poland
[email protected],
Ukrain is a semisynthetic drug with immunomodulatory prperties, derived from Chelidonium majus L. alkaloids and thiophosphoric acid. Previously studies showed
that Ukrain can activate Cytochrome P-450 (CYP2E1). In
the present study Ukrain was used to evaluate the effect on
NADPH-cytochrome P-450 reductase activity. This enzyme was obtained from human hearts, lungs and livers
post mortem. The causes of death were traffic accidents.
The activity of NADPH-cytochrome P-450 reductase was
evaluated by measuring the rate of cytochrome c reduction
at 550 nm at 37 oC (pH=7.4). First, activity of the enzymes
alone and subsequently, activity of NADPH-cytochrome
P-450 was measured in the presence of different concentrations of Ukrain (1, 2, 5 µl in total volume of 1000 µl).
Our study shows that Ukrain increased the NADPH-cytochrome P-450 reductase activity in the heart, lungs and liver at all investigated doses. In all cases the activity was
enhanced with the rise of given concentrations. The rises
strength of enzyme activity caused by Ukrain were in the
following order: heart>liver>lung.
TP06
THE INTERACTION BETWEEN
THE ANTICANCER DRUG
ELLIPTICINE AND
CYTOCHROMES P450 DICTATES
ITS PHARMACOLOGICAL
EFFICIENCIES
MARIE STIBOROVÁa, JAN SEJBALb,
DAGMAR AIMOVÁa, TEREZA DLOUHÁa,
LUCIE BO¤EK-DOHALSKÁa,
KRISTINA FORSTEROVÁa, JITKA POLJAKOVÁa,
MICHAELA ZACHOVÁa,
MARTINA STIBOROVÁ-RUPERTOVÁa,
KATE¤INA KUKAâKOVÁa, ANNA B¤EZINOVÁa,
JAN KUâKAa, and EVA FREIc
a
Department of Biochemistry and bDepartment of Organic
Chemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2, The Czech Republic, cDepartment of Molecular Toxicology, German Cancer Research
Center, Im Neuenheimer Feld 280, 69 120 Heidelberg,
Germany
Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole)
and several of its derivatives isolated from Apocyanaceae
plants are alkaloids exhibiting significant antineoplastic
and anti-HIV activities. We described that cytochrome
P450 (CYP)-dependent metabolism of ellipticine leads to
activation of this agent to more efficient metabolite(s) forming DNA adducts1,2. This shows the potential importance
of several CYPs in producing these more active antitumor
metabolite(s). The pattern of ellipticine metabolites formed
by CYPs has not been characterized so far. Therefore, in
the present work we investigated the metabolites of this anticancer drugs mediated by CYP catalyzed reactions. Moreover, ellipticine was also found to be an inhibitor of cytochromes P450, frequently used as a „selective“ inhibitor
of CYP1A1. Although ellipticine was reported to be a selective and strong inhibitor of CYP1A1, we found that its
inhibitory potential is not specific.
Ellipticine binds with greater affinity to CYPs than
most other compounds known to interact with CYPs. Ellipticine interacts with CYPs exhibiting a reverse type
I binding spectrum (λmax 430 nm) in microsomes isolated
from livers of uninduced rats and in those of rats pre-treated with β-naphthoflavone (β-NF), pregnenolon 16α-carbonitril (PCN), phenobarbital (PB) or ethanol with apparent
dissociation constants (Ks) of 1.44, 2.38, 5.71, 16.63 and
2.26 µM, respectively. Five metabolites containing an oxygen atom in their molecules (M1-M5) are generated from
ellipticine by CYPs; two of them were identified to be 9hydroxy- and 7-hydroxyellipticines. Both these metabolites are predominantly generated by CYP1A1/2. The M5
metabolite was identified to be N(2)-oxide of ellipticine,
while the structures of M2 and M3 metabolites have not yet
S117
been exactly characterized. All three ellipticine metabolites (M2, M3, M5) are mainly formed by CYP3A.
Control, β-NF-, PB-, ethanol- and PCN-induced microsomal CYP activities are inhibited by ellipticine. The degrees of CYP inhibition by ellipticine were quantified.
6β–hydroxylation of progesterone (Ki=0.021 µM) was most
effectively inhibited by ellipticine, followed by
CYP1A1/2-dependent EROD activity (Ki=0.038 µM) and
CYP2B-mediated PROD activity (Ki=0.05 µM). Elliptine
acts as a non-competitive or mixed-type inhibitor of these
enzymes. Lower, but measurable, inhibition was detected
for 1’ hydroxylation of bufurarol, 21-hydroxylation of progesterone and 6-hydroxylation of chlorzoxazone catalyzed
by CYP2D, CYP2C and CYP2E1, respectively.
The results indicate that the extent of inhibition by ellipticine might be explained by its differential potency to bind to
individual CYP or its inhibition of another enzyme of the microsomal system, NADPH:CYP reductase. The mechanism
responsible for CYP inhibition by ellipticine is discussed.
REFERENCES
1. Stiborová M., Bieler C.A., Wiessler M., Frei E.: Biochem. Pharmacol. 62, 1675 (2001).
2. Frei E., Bieler C.A., Arlt V., Wiessler M., Stiborová M.:
Biochem. Pharmacol, 64, 289 (2002).
Supported by Grant Agency of the Czech Republic
(grant 203/01/0996) and the Ministry of Education of the
Czech Republic (grant MSM 1131 00001).
TP07
NON-AROMATIC
HYDROXYLATION OF
BUPIVACAINE IN MAN
A. KUMARa, B. HOFTEb, R. LEDGERa,
and U. TJADENb
a
School of Pharmacy, Univ. of Otago, Dunedin, New Zealand, bAnalytical Chem, Gorlaeus Laboratories, Univ. Leiden, Holland, [email protected]
Urine from patients receiving continuous epidural infusions of bupivacaine were investigated for evidence of
non-aromatic hydroxylation. Urine extracts were analysed
by chiral LC-MS (liquid chromatography with mass spectrometric detection) on a a1-glycoprotein column at pH 7
using a volatile buffer of hydroxylamine acetate in aqueous
2-propanol (3% v/v). (S)-Bupivacaine was more extensively metabolised than (R)-bupivacaine and dealkylation
was the predominant pathway. However, at least 18 oxygenated metabolites of bupivacaine were detected by collision induced dissociation. Equal numbers of mono- and dihydroxybupivacaines were excreted of which there were
equal numbers of aromatic and non-aromatic hydroxylated
metabolites. There was no evidence to suggest that any one
of these was (S)-4’-hydroxybupivacaine, 2’-hydroxymethyl-bupivacaine, 3’-hydroxy-2’,6’-pipecoloxylidide or
a d-lactam. The metabolite previously identified as (S)-4’hydroxybupivacaine is not hydroxylated on the aromatic
ring. There was a wide inter-patient variation. The mean
rate of excretion of (S)-3’-hydroxylbupivacaine relative to
the (R)-desbutylbupivacaine excreted from three patients
(1.76 0.48) was greater than for seven patients (0.19
0.09). Conversely, the rate of excretion of (S)-desbutylbupivacaine relative to (R)-desbutylbupivacaine from the
group of three patients was 0.32 0.05 while for the group
of seven it was 1.28 0.09. Studies are proceeding to identify the CYP isoforms responsible for the hydroxylations.
TP08
not reduce successfully other activities involving
CYP2E1.
Our results suggest further participation of CYP2A6
and CYP2C19 enzymes in p-nitrophenol hydroxylation.
Further aim of our study was to evaluate the selectivity of
p-nitrophenol hydroxylase inhibitors towards P450 enzymes.2 The effects of antifungals: bifonazole, econazole,
clotrimazole, ketoconazole, miconazole; CNS-active
drugs: chlorpromazine, desipramine, fluphenazine, thioridazine; and the non-steroidal anti-inflammatory drug: diclofenac were investigated on the enzyme activities selective for CYP2A6, CYP2C9, CYP2C19, CYP2E1 and
CYP3A4. None of the drugs could be considered as a potent inhibitor of CYP2E1 (Table). Strong inhibition was
observed for CYP3A4 by antifungals with IC 50 values in
submicromolar range. However, ketoconazole was the only
imidazole derivative that could be considered as a selective
inhibitor of CYP3A4. The investigated CNS-active drugs
were not found to be potent inhibitors of CYP2A6,
CYP2C9, CYP2C19, CYP2E1 and CYP3A4. Diclofenac
efficiently inhibited CYP2C9 and to a less extent CYP3A4
enzyme.
INHIBITION OF P450
ENZYMES PARTICIPATING IN
P-NITROPHENOL HYDROXYLATION BY DRUGS KNOWN AS
CYP2E1 INHIBITORS
REFERENCES
KATALIN MONOSTORY, ESZTER HAZAI, and
LÁSZLÓ VERECZKEY
1. Koop D.R., Laethem C.L., Tierney D.J.: Drug Metab.
Rev. 20, 541 (1989).
2. Tassaneeyakul W., Birkett D.J., Miners J.O.: Xenobiotica 28, 293 (1998).
Chemical Research Center, Hungarian Academy of Sciences, Pusztaszeri 59-67, H-1525 Budapest, Hungary
[email protected]
p-Nitrophenol hydroxylation is widely used as a probe
for microsomal CYP2E1.1 Several drugs are known as
CYP2E1 inhibitors because of their capability to inhibit pnitrophenol hydroxylation.2 However, some of them did
Table 1.: IC50 values of the drugs towards selective enzyme activities.
The incubations were carried out using human liver microsomes of three different donors. IC50 value is expressed as µM. Values
represent mean + S.D.
Bifonazole
Clotrimazole
Econazole
p-nitrophenol
hydroxylation
Coumarin
7-hydroxylation
CYP2A6
Tolbutamide
4-hydroxylation
CYP2C9
(S)-mephenytoin
4’-hydroxylation
CYP2C19
Chlorzoxazone
6-hydroxylation
CYP2E1
Nifedipine
oxidation
CYP3A4
49.2±44.04
0.91±0.27
2.75±1.71
2.17±1.36
73.8±21.32
0.67±0.42
>250
11.25±5.86
5.91±1.65
73.2±46.41
>250
0.07±0.03
166.7±75.75
180.8±75.02
5.03±2.98
0.84±0.14
129.4±35.98
0.41±0.35
Miconazole
>250
132.8±110.86
5.41±3.52
1.78±0.84
>250
2.04±0.60
Ketoconazole
>250
>250
52.1±30.21
122.1±77.42
>250
0.40±0.16
Chlorpromazine
>1000
535.7±137.66
797.3±247.12
338.2±238.76
~1000
798.1±119.50
Desipramine
~1000
830.3±154.06
>1000
546.1±417.78
719.9±191.55
339.7±71.4
Fluphenazine
>1000
477.4±158.12
398.1±258.25
528.3±409.27
735.2±48.43
441.2±241.00
Thioridazine
>1000
405.2±45.48
734.0±119.52
252.1±181.75
>1000
392.7±25.96
Diclofenac
>1000
>1000
69.7±33.08
>1000
>1000
698.4±214.5
S118
TP09
dibenzylfluoresceine metabolism was detected. To our
knowledge it is the first evidence for the stimulation of the
enzymatic activity of a steroidogenic CYP19. Thus, flavonoids tested in this study can exert a rather complex effect
on CYPs, either inhibit or activate human cytochromes
P450 depending upon their structures, concentrations, and
experimental conditions.
CYP19 ACTIVITY
MODULATION: SMALL
CHANGE OF FLAVONOID
STRUCTURE RESULTS IN
INHIBITOR-STIMULATOR
TRANSITION
DANA PROVAZNÍKOVÁ, PETR HODEK,
STANISLAV SMRâEK, BARBORA I·TVÁNKOVÁ, and
MARIE STIBOROVÁ
University, Albertov 2030, 128 40 Prague 2, The Czech Republic, [email protected]
An inhibitory capacity of flavonoids with respect to
CYP activities has been extensively studied because of
their potential use as agents blocking the initiation of carcinogenesis. Synthetic and naturally occurring flavonoids
are effective inhibitors of at least four CYPs metabolizing
xenobiotics: CYP1A1, 1A2, 1B1 and 3A4. On contrary,
certain metabolic activities of CYP3A4 and 1A2 were also
stimulated by several compounds of flavonoid structure.
For example, synthetic 7,8-benzoflavone (ANF) is an inhibitor of human CYP1A1 and 1A2, but an activator of
CYP3A4. CYP19, aromatase, is another member of cytochrome P450 enzyme super-family that is effectively inhibited by flavonoids since these compounds resemble the
skeleton of steroids. This enzyme catalyzes a unique reaction, aromatization of the A ring of male sex steroids resulting in estrogens. Since estrogens are known cell proliferators and their metabolites, catechols, are carcinogens,
a local expression of aromatase is suggested to be closely
connected with tumor initiation, promotion and progression. Hence, aromatase inhibitors are used as pharmaceuticals in the prevention and/or treatment of especially hormone-dependent breast and prostate cancers. The most effective aromatase inhibitor of flavonoid structure are ANF
and its 9-hydroxy-derivative, showing IC50 values comparable to a strong steroid aromatase inhibitor, 4-hydroxyandrostendione. The presence of C4 oxo-group of the flavonoid skeleton, which is likely approaching the heme iron
(based on models), seems to be crucial for inhibition. One
can expect that the substitution of oxygen for sulphur in
this position will result in a stronger binding of the compound to aromatase increasing its inhibition capability.
Surprisingly, a thio-analogue of ANF (SANF) formation of
estrone from androstendione (160% of control) in human
placental microsomes. The same stimulatory effect of
SANF on CYP19 was determined with its non-physiological substrates, 7-ethoxycoumarin and dibenzylfluoresceine, in microsomes from baculovirus-infected insect cells
containing co-expressed human CYP19 and NADPH:cytochrome P450 reductase (150% of control). In addition, in
human placental microsomes almost 200% stimulation of
S119
TP10
POTENT MECHANISM-BASED
INHIBITION OF HUMAN CYP2B6
BY CLOPIDOGREL AND
TICLOPIDINE
TANJA RICHTER, KATHRIN KLEIN,
THOMAS E. MÜRDTER, MATTHIAS SCHWAB,
MICHEL EICHELBAUM, and ULRICH M. ZANGER
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstr. 112, 70376 Stuttgart, Germany,
[email protected]
Thrombolytic drugs are widely used in patients with
coronary heart disease (CHD). These include the thienopyridine derivates ticlopidine and clopidogrel, which are
inhibitors of ADP-induced platelet aggregation. Since
clopidogrel shows a more rapid onset of action and a lower incidence of adverse effects compared to ticlopidine,
it is more frequently used today 1. Clopidogrel is metabolized to its antithrombotic agent by CYP3A4 2 and a recently published clinical study demonstrated an inhibitory effect of the CYP3A4 substrate atorvastatin on platelet aggregation 3. The aim of this study was to investigate the interaction potential of clopidogrel towards cytochrome P450 enzymes. Using human liver microsomes
and recombinantly expressed enzymes we found that
both clopidogrel and ticlopidine strongly inhibit
CYP2B6 in a time-and concentration-dependent manner
with IC50 values of 0.8 µM and 1.1 µM, respectively. As
shown by dialysis experiments, inhibition was irreversible and dependent on the presence of NADPH, strongly
suggesting a mechanism-based mode of action for both
clopidogrel and ticlopidine. A high specifity of both substances for CYP2B6 was demonstrated by analyzing enzyme activities selective for other human liver enzymes
including CYP1A2, CYP2E1, CYP2A6, CYP3A4,
CYP2C8, CYP2C9 and CYP2C19, although the latter
isozyme was also inhibited to some extent, as has been
reported in an earlier study 4. These results suggest the
possibility of drug interactions between these two drugs
and other drug-substrates of CYP2B6. Clopidogrel in
particular may prove to be a useful in-vitro inhibitor
probe for CYP2B6.
Supported by the Deutsche Forschungsgemeinschaft
(grant ZA 245/2-1) and by the Robert Bosch Foundation
Stuttgart Germany.
REFERENCES
1. Kam P.C. and Nethery C.M.: Anaesthesia 58, 28 (2003)
2. Clarke T.A. and Waskell L.A. Drug Metab Dispos 31,
53 ( 2002)
3. Lau W.C.et al.: Circulation 107, 32 ( 2003)
4. Ha-Duong N.T.et al.: Biochemistry 40, 12112 (2001)
TP11
CYP17 CATALYTIC
IRON-OXYGEN SPECIES
DICTATED BY CYTOCHROME B5
M.E., Akhtar M.: Biochemistry 34, 14104 (1995).
2 Lee-Robichaud P., Shyadehi A.Z., Akhtar M.E., Akhtar
M.: Biochem J. 308, 901 (1995).
3 Katagiri M., Kagawa N., Waterman M.R.: Arch. Biochem. Biophys. 317, 343 (1995).
4 Ravichandran K.G., Boodupalli S.S., Hasemann C.A.,
Peterson J.A., Deisenhofer J.: Science 261, 731 (1993).
TP12
FARADAIC BIOELECTROCHEMISTRY FOR THE DRUG
METABOLISM ASSAY
VICTORIA V. SHUMYANTSEVA,
TATIANA V. BULKO, and
ALEXANDER I. ARCHAKOV
PETER LEE-ROBICHAUDa, QAISER SHEIKHa, and
MUHAMMAD AKHTARb
119121 Moscow Russia, [email protected]
a
Centre for Chemical Biology, Dept. of Chemistry, University
of Sheffield, Sheffield, S3 7HF, U.K.; bDept. of Biochemistry,
University of Southampton, Southampton, SO16 7PX, U.K.
[email protected]
Microsomal cytochrome P450 17α-hydroxylase-17,20lyase (CYP17) has the ability to catalyse 17α-hydroxylation
and acyl-carbon bond cleavage of the C21 progestogens. We
have proposed that the hydroxylation reaction is catalysed by
a heam-iron-monooxygen radical species whereas the cleavage process by a heam-iron-peroxide anion1. However, for
the human isoform of CYP17 to catalyse the cleavage reaction a third protein, cytochrome b5 (b5), is required2,3. It is proposed that b5 promotes a conformational change in CYP17
which culminates in the optimal juxtaposition of the haemiron-peroxide anion with respect to the C-20 carbonyl of the
substrate2. The ensuing nucleophilic attack at the carbonyl
group then produces a tetrahedral intermediate that follows
a side chain cleavage path. Using the X-ray structural information for P450BM-34 and linear amino acid sequence alignments, the most likely docking site on CYP17 for b5 was
identified. Surface-exposed cationic residues within this putative b5 docking site, that are proposed to form electrostatic
interactions with anionic charges on b5, were mutated to remove their charge and the expressed mutant proteins purified. The mutant CYP17 enzymes were subjected to kinetic
analysis, utilizing the novel properties of three different substrates to probe b5-CYP17 interaction. The results are presented and the determined metabolic profile is consistent with
our proposal. Part of the docking interface between CYP17
and b5 is putatively mapped onto the surface of a computer
generated 3D homology model of CYP17.
REFERENCES
1 Lee-Robichaud P., Shyadehi A.Z., Wright J.N., Akhtar
S120
Cytochromes P450 (CYPs) catalyze the oxidative metabolims of a wide variety of exogenous and endogenous
chemicals such as drugs and carcinogens. Potential applications range from the use of cytochromes as drug targets
to the use of these enzymes as tool to predict drug-drug interactions in humans. The industrial application of cytochrome P450-containing systems is tightly bound with the
usage of alternative electron sources for P450s’ reduction.
Cytochrome P450-based electrochemical devices allowed
to construct sensor systems for screening a wide variety of
drugs on the possibility of drug-drug interactions via CYP,
namely high throughput screening. Thick film screen-printed graphite electrodes served as working electrodes. Immobilized flavocytochromes and cytochromes P450 layers
were prepared by cross-linking of enzyme with glutaric aldehyde or with entrapment in the [3-(2,3-epoxypropoxy)propyl]-trimethoxysilane matrix. Sol-gel matrices were effectively used for the entrapment of CYPs. Voltammetric
techniques were used to investigate the kinetics of P450 redox reactions. Kinetic parameters of redox system were
calculated with algebraic transformation of the MichelisMenten equation known as the Hill equation and with the
electrochemical Lineweaver-Burk plots. The catalytic activity of electrochemical systems based on CYP 2B4, CYP
1A2 and CYP11A1 (P450scc) were characterized with the
help of kinetic parameters of enzyme electrodes, such as
apparent Michaelis-Menten constant, catalytic constant, reaction rates (Kmapp, kcat, Vmax). The ratio of the catalytic current to the diffusion controlled current Icat/Id was has been taken as a measure of catalytic efficiency of the electrochemical system. From the integration of peaks in the cyclic
voltammograms the apparent surface coverage of the electroactive cytochrome P4502B4 and P450scc was calculated.
With the help of electrochemical artificial P450-based devices it is possible to analyse potential substrates or inhibi-
tors of P450 investigated. The catalytic current Icat of the
enzyme electrode changes with the concentration of substrate. Thus Icat and steady state current response of P450electrodes upon substrates addition could be used to measure drug (substrate) concentration. The inhibitory effects
of some chemicals on the catalytic current of cytochrome
P450-based bioelectrodes can be estimated. Enzyme electrodes described provide approach for the fabrication of
specific, portable and low cost disposable cytochrome
P450-based amperometric or potentiometric biosensors.
This work was supported by the State Program of Russian Ministry of Sciences and Technology and by RFBR
grants
REFERENCES
1. Shumyantseva V. V., Bulko T. V., Schmid R. D., Nicolini
C., Archakov A. I.. J. Inorg. Biochem., 87, 185 (2001)
2. Shumyantseva V. V., Bulko T. V., Schmid R. D., Archakov A. I. Biosensors and Bioelectronics, 17, 233
(2002).
3. Shumyantseva V. V., Bulko T. V., Archakov A. I. 14th Int.
Symposium on Microsomes and Drug Oxidation. 2002,
Sapporo, Japan, Book of Abstracts, 177, P26-C-54.
TP13
STRUCTURAL AND FUNCTIONAL CHARACTERIZATIONS
OF PUTIDAREDOXIN-INDUCED
STRUCTURAL CHANGES
IN CYTOCHROME P450CAM
TAKEHIKO TOSHAa, SHINGO NAGANOb,
JASON K. YANOb, SHIRO YOSHIOKAa,
HARUYUKI HARADAa, SATOSHI TAKAHASHIa,
KOICHIRO ISHIMORIa, THOMAS L. POULOSb
and ISAO MORISHIMAa
Even in the absence of Pdx, the 1H NMR signals from
the β-proton of the axial Cys and the substrate, d-camphor,
were up-field shifted for carbonmonoxy L358P, as observed for wild type P450cam in the presence of Pdx2, showing that both d-camphor and the axial Cys move toward
to the heme iron in L358P. The structure of L358P in the
absence of Pdx, therefore, corresponds to that of wild type
P450cam in the P450cam-Pdx complex, which enables us
to gain some insights into the Pdx-induced structural changes in P450cam without the crystallization of the
P450cam-Pdx complex. The X-ray crystal structure of carbonmonoxy L358P showed the tilting of the heme plane,
leading to the positional change of Arg112 at the putative
Pdx binding site, through the hydrogen bond between
Arg112 and the heme propionate. We can conclude that
the binding of Pdx to P450cam induces the tilting of the
heme plane by perturbing the hydrogen bond between the
heme propionate and Arg112, resulting in the approach of
d-camphor toward the heme iron. In order to clarify the
functional significance of the Pdx-induced structural changes in P450cam, we also investigated the reactivities of
L358P with electron donors. Because the Pdx-induced
structural changes in P450cam are crucial for the electron
transfer reaction, we hypothesize that L358P would exhibit
enhanced reactivities with the non-physiological electron
donors that cannot donate the electron to the oxygen adduct
of wild type P450cam but have the lower redox potential
than Pdx. As we expected, the mixing of oxy-L358P with
the non-physiological electron donor such as dithionite in
the presence of d-camphor produced significant amounts of
the hydroxylation product, 5-exo-hydroxycamphor. Considering the lowered redox potential of L358P1, we, therefore, propose that the Pdx-induced structural changes in
P450cam would facilitate the electron transfer to oxyP450cam by regulating the electronic coupling and/or reorganization energy.
REFERENCES
a
Department of Molecular Engineering, Graduate School
of Engineering, Kyoto University, bDepartment of Molecular Biology & Biochemistry, University of California,
Irvine, [email protected]
Cytochrome P450cam (P450cam) catalyzes hydroxylation of d-camphor by using two electrons from its redox
partner, putidaredoxin (Pdx). In addition to being an electron shuttle to P450cam, Pdx acts as a conformational
effector of P450cam, which is believed to be crucial
for the enzymatic activity of P450cam. However, the detailed mechanism of the Pdx-induced structural changes in
P450cam is not clear owing to the lack of the crystal structure
of the P450cam-Pdx complex. Here, to characterize the Pdxinduced structural changes in P450cam and their functional
role in the catalytic cycle, we utilized the Leu358➝Pro
(L358P) mutant1 as a model for Pdx bound P450cam.
S121
1. Yoshioka et al. J. Inorg. Biochem. 2000, 81, 141.
2. Tosha et al. Submitted for publication.
3. Lipscomb et al. J. Biol. Chem. 1976, 251, 1116.
TP14
REFERENCES
CHIRAL ASPECTS OF
REDUCTION METABOLISM OF
FLOBUFEN IN GUINEA PIGS
IN VIVO
1. Fujimoto R.: Curr. Opin. CPNS Invest. Drugs 1, 142
(1999)
2. Kvasniãková E., Szotáková B., Wsól V. et al.: Exp. Toxicol. Pathol. 51, 352 (1999)
3. Trejtnar F., Wsól V., Szotáková B. et al.: Chirality 11,
781 (1999)
4. Wsól V., Král R., Skálová L. et al.: Chirality 13, 754
(2001)
FRANTI·EK TREJTNARa, RADIM KRÁLb,
VLADIMÍR WSÓLb, PETR PÁVEKa, and
EVA KVASNIâKOVÁb
a
Department of Pharmacology and bDepartment of Biochemical Sciences, Faculty of Pharmacy, Charles University,
Hradec Králové, Czech Republic
Metabolic transformation of flobufen [4-(2’,4’-difluoro-biphenyl-4-yl)-2-methyl-4-oxobutanoic acid], a chiral
non-steroidal antiinflammatory agent1, includes its conversion to a reduced metabolite2. Reduced flobufen
[4-(2’,4-difluoro-biphenyl-4-yl)-4-hydroxy-2-methylbutanoic acid] is the major metabolite generated by the subcellular fractions. It was detected upon incubation with liver microsomes from several animal species. Maximum
yield of reduced flobufen (dihydroflobufen) was found after anaerobic incubation with NADPH. This finding combined with the knowledge of subcellular distribution of
enzymes suggested that metabolite formation depends on
the activity of microsomal reductase, cytochrome P-4502.
Flobufen possesses an α chiral carbon atom next to the
carboxyl group and its enantioselective bioelimination has
been proved in rats3. Reduction of flobufen resulted in
another center of chirality in the molecule and four stereoisomeric dihydrometabolites can be formed from two parent flobufen enantiomers4. We studied the metabolism of
flobufen to its reduced metabolite in male guinea pigs
in vivo. Especially, chiral aspects of the reduction metabolic process were analyzed. Flobufen was administered as
rac-flobufen (R/S-enantiomer = 50/50) p.o. in a dose of
10 mg/kg. The drug was metabolized in a significant rate
in the guinea pig. All four stereoisomers of dihydrometabolite of flobufen were detected in guinea pig plasma. However, the found concentrations were very different. The
highest levels in plasma showed (2R;4R)-dihydroflobufen,
the lowest levels were found in case of (2S;4R)-dihydroflobufen. The concentration ratio of these two circulating
diastereoisomers in guinea pig plasma reached up to more
than one order of magnitude. The main product of biotransformation of flobufen found in guinea pig plasma was
nonchiral metabolite 17203 [4-(2,4-difluorophenyl)-phenylacetic acid]. The metabolite reached plasma levels
comparable to parent flobufen enantiomers. However, metabolite 17203 is formed from reduced flobufen2. It means
that the stereospecific reduction of flobufen by microsomal reductases seems to be the key step in flobufen metabolism in guinea pigs.
This work was supported by the Research project
LN00B125 of the Czech Ministry of Education.
S122
TP15
MULTIPLE P450 SUBSTRATES IN
SINGLE RUN: RAPID, COMPREHENSIVE AND CLINICALLY
RELEVANT IN VITRO
METABOLISM TEST
MIIA TURPEINENa, JOUKO UUSITALOa,b,
JORMA JALONENb, and OLAVI PELKONENa
a
Department of Pharmacology and Toxicology and bDepartment of Chemistry, University of Oulu, P.O.BOX 5000,
FIN-90014 University of Oulu, Finland,
[email protected]
The aim of this study was to develop a global in vitro
screening test utilizing N-in-1 approach, wihich could answer the following questions: what is metabolic stability of
a new chemical entity (NCE) studied, which enzyme(s)
metabolise NCE and which enzyme(s) is/are target(s) for
further interaction studies? By using appropriate concentrations and incubations this test should reveal affinities of
NCE for different enzymes, kinetic properties of NCE
(Km, Vmax) for each metabolising enzyme and prediction
of in vivo behaviour.
A cocktail consisting of ten CYP-specific probes: melatonin (CYP1A2), coumarin (2A6), bupropion (2B6),
amodiaquine (2C8), tolbutamide (2C9), omeprazole
(2C19), dextromethorphan (2D6), chlorzoxazone (2E1),
midazolam and testosterone (3A4) was incubated in a pool
of human liver microsomes in the presence of NADPH.
Analysis of samples was performed using LC/TOF-MS (total analysis time 20 minutes, 0.1%formic acid-methanol
gradient, flow rate 0.25 ml/min). No post-incubation extraction or concentration procedures were needed.
Validation of the method has been performed with diagnostic CYP-specific inhibitors: fluvoxamine (CYP1A2),
tranylcypromine (2A6), ticlopidine (2B6), quercetin (2C8),
sulphaphenazole (2C9), fluconazole (2C19), quinidine
(2D6), pyridine (2E1) and ketoconazole (3A4). The results
were in good accordance with literature and our previous
studies.
In addition, with liver microsomes this test has been succesfully applied to cDNA-expressed CYP enzymes,
mouse hepatocytes and permanent cell lines. Validation of
the method with NCEs with known metabolic profiles is
under preparation.
TP16
IMMOBILISATION OF P450 BM-3
AND AN NADP+ COFACTOR
RECYCLING SYSTEM:
TOWARDS A TECHNICAL
APPLICATION
STEFFEN C. MAURER, ROLF D. SCHMID, and
VLADA URLACHER
Institute of Technical Biochemistry, Stuttgart University,
Allmandring 31, 70569 Stuttgart, Germany,
[email protected]
P450 BM-3 (CYP102), which was originally cloned
from Bacillus megaterium, is a 119 kDa natural fusion protein composed of a heme-containing monooxygenase domain and an FAD- and FMN-containing reductase domain1,2. The turnover frequencies determined for P450 BM3 (>1000 min-1) are, to date, among the highest reported for
a P450 monooxygenase. The natural substrates of P450
BM-3 are long chain fatty acids (C12 to C20). Artificial mutants of P450 BM-3 also hydroxylate non-natural substrates like short-chain fatty acids (C8 to C10)3, indole, polycyclic aromatic hydrocarbons and alkanes4. These hydroxylation reactions can lead to regio- and/or stereochemically pure compounds. As regio- and stereospecific oxidation of aliphatic carbons is a reaction, which is difficult to
perform by means of classical organic synthesis, the biocatalysis by this monooxygenase is a promising option, especially with regard to fine chemistry. Key problems preventing the use of P450s in biotechnology are relative low stability and the supply of electrons to the heme iron. In vivo,
the electrons are supplied by NAD(P)H, which is however
far too expensive to be used in technical applications. To
render P450 BM-3 more suitable for industrial biocatalysis, the immobilisation of P450 BM-3 (CYP 102) from Bacillus megaterium in a sol-gel matrix was combined with
a cofactor recycling system based on NADP+-dependent
formate dehydrogenase (EC 1.2.1.2) from Pseudomonas
sp. 101 and tested for practical applicability5.
REFERENCES
1.
2.
3.
4.
5.
Wen P., Fulco A.J.: Biol. Chem., 262, 6676 (1987)
Fulco A.J., Ruettinger R.T. Life Sci., 40, 1769 (1987)
Ost T. W. et al. FEBS Lett., 486, 173 (2000).
Appel D et al.. Biotechnol., 88, 167 (2001).
Maurer S.C., Schulze H., Schmid R.D., Urlacher V.
Adv.Syn.Biocat., (2003) (in press)
S123
TP17
HETEROLOGOUS EXPRESSION
OF FULL-LENGTH NADH-CYTOCHROME B5 REDUCTASE AND
ITS FUSION PROTEIN WITH
CYTOCHROME B5
ANDREI A. GILEP, VADIM V. KOKHANOVSKI,
SERGEY A. USANOV
Institute of Bioorganic Chemistry National Academy of
Sciences of Belarus, Minsk, Belarus
[email protected]
NADH-cytochrome b5 reductase is essential human liver and intestine enzyme, which involved in cytochrome b 5
reduction and participates in metabolism of a large number
of chemicals of different structure, functions and size. The
enzyme exists in two forms: a membrane-bound form that
is comprised of both a hydrophobic membrane-anchoring
region and a hydrophilic, or catalytic domain, and a soluble
form which contains only a hydrophilic domain. Recently,
soluble NADH-cytochrome b5 reductase has been expressed in E. coli, purified and intensively studied. However,
the role of hydrophobic N-terminal sequence of full-length
NADH-cytochrome b5 reductase, that anchors flavoprotein
in the membrane, remains unknown.
The aim of the present work is to develop efficient expression system for full-length NADH-cytochrome b5 reductase. To solve this problem we constructed several plasmids containing cDNA for full-length NADH-cytochrome
b5 reductase under Tac and T7 promotors. We also introduced the His-tag fragment to the C-terminal sequence of flavoprotein to improve purification procedure. The expression level of NADH-cytochrome b5 reductase under T7
promotor proved to be significantly higher than that of under Tac promotor. The effective purification procedure for
recombinant NADH-cytochrome b5 reductase based on
using immobilized recombinant cytochrome b5 has been
developed.
We also constructed, expressed and purified fusion protein between full-length NADH-cytochrome b5 reductase
and cytochrome b5 and showed that fusion protein contains
both heme and FAD prosthetic groups and able to intra-molecular electron transfer.
TP18
OVEREXPRESSION OF
FULL-LENGTH OUTER
MITOCHONDRIAL
CYTOCHROME B5 IN E. COLI
TP19
ENGINEERING OF
PROTEOLYTICALLY STABLE
NADPH-CYTOCHROME P450
REDUCTASE
ANDREI A. GILEPa, GENNADY V. SERGEEVa,
AKIO ITOb, and SERGEY A. USANOVa
ANDREI A. GILEPa, TATYANA A. BONINAa,
RONALD W. ESTABROOKb, and SERGEY A. USANOVa
a
Institute of Bioorganic Chemistry National Academy
of Sciences, Minsk, Belarus; bDepartment of Chemistry,
Kyushu University, Fukuoka, Japan
[email protected]
a
Institute of Bioorganic Chemistry NASB, Kuprevicha 5/2,
220141 Minsk, Belarus,; bDepartment of Biochemistry,
UT SWMED, TX, USA, [email protected]
Outer mitochondrial membrane-bound cytochrome b5,
the isoform of microsomal cytochrome b5, is a typical tailanchored hemeprotein of outer mitochondrial membrane.
Both mitochondrial and microsomal cytochromes b5 have
similar organization. They have hydrophilic N-terminal
domain, which preserves spectral properties of cytochrome
b5 and C-terminal hydrophobic domain, which is responsible for interaction with membrane and functions as reporter for insertion hemeprotein to membrane as a tailedanchored protein. The hydrophilic N-terminal domain of
cytochrome b5 consists of about 100 amino acids, contains
heme and participates in electron transfer reactions. The Nterminal sequence of outer mitochondrial cytochrome b5
contains extra 10 amino acids the role of which is unknown. Hydrophilic sequences of cytochrome b5 and outer
mitochondrial isoform of hemeprotein show 70% of identity. The hydrophobic C-terminal domain of cytochrome b5
contains three subdomains: a. hydrophilic linker; b. intrinsic hydrophobic sequence (about 20 amino acids), which is
embedded to the lipid bilayer; c. hydrophilic tail which
contains information on protein translocation. The role of
hydrophobic domain of outer mitochondrial cytochrome b5
remains unknown.
The aim of the present work is to develop an efficient
expression system for full-length outer mitochondrial cytochrome b 5 and to study the functional role of the hydrophobic C-terminal sequence. Surprisingly, our efficient
expression system developed previously for microsomal
cytochrome b 5 based on pCWori + vector and Tac promotor, did not result in expression of outer mitochondrial
cytochrome b 5. Removal of extra N-terminal amino acid
residues did not improve the expression. However, cloning the cDNA coding outer mitochondrial cytochrome
b5 under T7 promoter (vector pT7) resulted in over expression of hemeprotein in some cases up to 6000 nmoles per l of culture. Recombinant full-length outer mitochondrial cytochrome b 5 and its N-terminal truncated
forms have been expressed in E. coli, purified to apparent homogeneity using metal-affinity chromatography
giving about 1.5-3.0 (mole of homogeneous full-length
outer mitochondrial cytochrome b 5 which was used to assess the effect on cytochrome P4503A4 and P45017 a catalyzed reactions.
S124
NADPH-cytochrome P450 reductase (NCPR) contains
hydrophobic N-terminal membrane-binding and hydrophilic catalytic domains. Truncated (trypsin-treated reductase), which does not contain the hydrophobic N-terminal
fragment, is still able to transfer electrons to cytochrome c and other artificial electron acceptor, but is no longer
capable to interact with cytochrome P450 and transfer electrons to it. The preferential site of tryptic attack on the rat
reductase was shown to be at the Lys56-Ile57 while the second recognition site for trypsin is Lys46-Lys47-Lys48 sequence that yields a small polypeptide (Lys46 or Lys47 through
Lys56). Moreover, Lys56-Ile57 is a site for spontaneous digestion of CPR by intercellular trypsin-like proteases,
which makes flavoprotein very unstable during purification
or expression in E. coli. The aim of this study is to evaluate the effect of a single amino acid substitution at position
Lys56 in attempt to create trypsin-insensitive flavoprotein.
For that we replaced Lys56 for Asn, expressed and purified
recombinant flavoprotein. Studies of kinetic mechanism of
NCPR mutant showed no significant differences between
0 min
R
1 min
M R
2 min
M R
5 min
M R M
10 min
R
M
A
105
75
50
R 0 1
B
RM
MM
minutes
minutes
5 10 30 0
1
5 10 30 K
105
75
50
Fig. 1. SDS-PAGE trypsin-treated purified (A) and membranebound reductases (B). Panel A: purified wild type (R) and mutant
(M) reductase. Panel B: membranes prepared from E. coli expressing the wild type (RM) and mutant form (MM) reductases.
wild type protein and Lys56Asn mutant. At the same time,
replacement of Lys56 makes the full-length flavoprotein
stable to spontaneous proteolysis. During prolong incubation or increased trypsin ratio, mutant form has the other limited proteolysis pattern, indicating the accessibility of the
second site. The membrane-bound mutant form is absolutely stable to trypsinolysis. These results let us to conclude
that the polypeptide fragments Lys46-Lys48 is localized in
membrane. Truncated form of mutant flavoprotein
(AA’s 46-676 of NCPR) unable transfer electrons to cytochrome P450c17.
TP20
C-TERMINAL RESIDUES OF
CYTOCHROME P450SCC ARE
REQUIRED FOR CORRECT
FOLDING, HEME BINDING
AND CATALYSIS
well as folding were not changed by C-terminal deletion.
Thus, C-terminal truncation analysis showed that this region of P450scc (9aa) is not critical for enzyme activity,
while removal 10 amino acids dramatically affects hemeprotein folding and heme binding. The role of C-terminal
region in oligomerization/aggregation of P450scc is
currently under investigation.
TP21
SPECTROSCOPIC STUDIES
OF CYTOCHROME P4503A4
INTERACTION WITH
FLAVONOIDS
MAXIM V. CHUDAEVa,
EGOR M. TCHERVIAKOVSKYb,
VLADIMIR P. KURCHENKOb, and
SERGEY A. USANOVa
a
Institute of Bioorganic Chemistry NASB, Kuprevicha 5,
220141 Minsk, Belarus,, bDepartment of Biochemisty, BSU,
Skaryny av., 4, Minsk, Belarus, [email protected]
GALINA I. LEPESHEVA,
NATALYA V. STRUSHKEVICH, TAMARA N. AZEVA,
and SERGEY A. USANOV
Institute of Bioorganic Chemistry National Academy of
Sciences of Belarus, 220141, Minsk, Kuprevicha 5, Belarus
[email protected]
To understand the functional role of C-terminal part of
mitochondrial cytochrome P450scc (P450scc) in folding
and heme binding, we used sequential amino acid deletion
strategy. A set of mutants lacking 2, 4, 7, 9 and 10 amino
acids from C-terminus of bovine P450scc were constructed, expressed and purified to compare with wild type protein. Removal of 10 amino acids from the C-terminus of
P450scc results in complete disappearence of spectrally detected P450 in E. coli culture as well as in the cell solubilizate. However, immunoblotting analysis indicates the
presence of apo-protein in cells. Alignment of P450’s from
different sources indicates that Arg472 might be the last residue in the protein sequence which is critical for P450scc
expression in catalytically active holo-form. In contrast,
protein shortened for 9 amino acids is fully competent to
support electron transfer in reconstitution system with
adrenodoxin and adrenodoxin reductase. Cytochrome
P450scc lacking 9 amino acids is expressed in E. coli rather poorly, but could be affinity-purified as enzymatically
active protein.
The expression level for mutants with deletion of 2 and
4 (nonconservative amino acids among mitochondrial
P450’s) is significantly higher as compared to the wild type
protein and these mutants reveal activity towards cholesterol side chain cleavage reaction and suitable for functional
and structural analysis. Deletion of seven amino acids
yields a hemeprotein with properties similar to full-length
P450scc. Kinetic and pattern of P450scc trypsinolysis as
S125
Flavonoids - low molecular weight phenols that are
found ubiquitously in the vascular plants. They have pronounced antioxidant properties and ability to influence on
Fig. 1. Titration curve of cytochrome P450 3A4 with quercetin.
variety of enzymes and cell receptors. Among all flavonoids, quercetin is belived to be the most consumed flavonoid. It is known as one of the most potent natural antioxidant and effective metal chelator. Quercetin inhibits activities of calcium phospholipid-dependent protein kinase,
phosphoinositol 3-kinase and DNA-topoisomerase, phospholipase A2. Currently there is growing evidence for the
inhibitory effect of some flavonoids including quercetin on
activity of cytochrome P4503A4. Cytochrome P4503A4 is
the major P450 isoenzyme in human liver and intestine,
which is involved in oxidations of a variety of xenobiotics
and chemicals.
The aim of present study is to elucidate weather flavonoids like quercetine directly interact with the active site of
cytochrome P450 and affect the reactions catalyzed by this
hemeprotein. To answer this question we expressed in
E. coli cytochrome P4503A4 as a His-tag derivative, purified recombinant hemeprotein using metal-affinity chromatography and used it in spectral binding studies.
The results of the spectral binding experiments indicate
that quercetin is able to interact with cytochrome P4503A4
and induce the typical type I spectral changes with absorbance maximum at 390 nm and minimum at 420 nm, indicating the change of the spin state of cytochrome P4503A4.
The Amax and Ks values calculated by nonlinear regression
of the plot ∆A versus S using Origin 5.0 software were
found to be 0,121 and 60 µM, respectively. The interaction
of cytochrome P4503A4 with other types of flavonoids and
their effects on cytochrome P4503A4 catalyzed reaction is
underway.
tion equal to the apparent Km value. In recombinant
CYP2D6, AMMC was found to be a good inhibitor of bufuralol 1’-hydroxylation and dextromethorphan O-demethylation with IC50 values of 2.3 and 2.2 µM, respectively.
Only moderate inhibition was observed by MAMC with
IC50 values of 33 and 21 µM, respectively. The interaction
between bufuralol and dextromethorphan was confirmed to
be competitive with Ki = 1.2 µM for dextromethorphan on
bufuralol 1’-hydroxylation and with Ki = 5 µM for bufuralol on dextromethorphan O-demethylation. However, in all
cases, the inhibitory potencies were decreased when human
liver microsomes were used in the study. Possible reasons
explaining the apparent discrepancy between the two enzyme systems will be discussed.
AMMC: 3-[2-(N,N-diethyl-N-methylammonium)-ethyl]-7-methoxy-4-methylcoumarin
MAMC: 7-methoxy-4-(aminomethyl)-coumarin
TP23
REDUCTION OF OXYFERROUS
CYTOCHROME P450 2B4 BY
CYTOCHROME P450
REDUCTASE
HAOMING ZHANGa, LARRY GRUENKEa,
DAVE ARSCOTTa, ANNA SHENb,
CHARLES KASPERb, and LUCY WASKELLa
a
TP22
University of Michigan and VA Medical Center, 2215 Fuller Rd., Ann Arbor, MI, [email protected], bMcArdle Laboratory, University of Wisconsin, Madison, Wisconsin
2D6-CATALYZED REACTIONS:
THE KINETIC INTERACTION
BETWEEN TWO SUBSTRATES
REGINA W. WANG, XIAOXIN CAI,
VARSHA DIDOLKAR, RICHARD W. EDOM,
RAYMOND EVERS, and DAVID C. EVANS
Department of Drug Metabolism, Merck Research Laboratories, Rahway, NJ, USA. [email protected]
More than 80 drugs in clinical use are metabolized by
polymorphic CYP2D6. It is important to evaluate whether
new chemical entities are potent inhibitors of this CYP isoform. Bufuralol and dextromethorphan have been used as
in vitro probes for inhibition studies. Recently, two fluorescent probes, AMMC and MAMC, were reported to be
good CYP2D6 substrates which can be used in a highthroughput screening format. In order to investigate in
vitro substrate-substrate interactions with CYP2D6, these
four compounds were studied in pairs using recombinant
CYP2D6 and human liver microsomes. For all studies, the
IC50 values were determined using the substrate concentra-
S126
The rate of electron transfer from cytochrome P450
reductase to oxyferrous cytochrome P450 2B4 (cyt P450),
i.e., donation of the second electron, has been measured
experimentally by using stopped-flow spectrophotometry
and a cyt P450 reductase containing a redox inactive FAD
analogue. This FAD analogue-containing cyt P450 reductase can be 2 electron reduced to the FMN hydroquinone
by dithionite. The FMNH2 oxidizes monophasically in the
presence of O2. Cyt c oxidizes both the native reductase
and the reductase reconstituted with the FAD analogue
with the same second order rate constants indicating that
the function of the FMN domain of the reductase is intact.
Since the reductase reconstituted with a FAD analogue donates a single electron from FMNH2, the spectral changes
it undergoes when it oxidizes to the semiquinone are easily
interpreted. This contrasts with what is observed in a 2e
reduced FAD-containing cyt P450 reductase where the
second electron is distributed between the FMN hydroquinone and FAD semiquinone according to their respective
potentials. In this instance the spectral changes occurring
on oxidation are complex and cannot be unambiguously interpreted. Ferrous cyt P450 2B4 and the 2 electron reduced
reconstituted cyt P450 reductase were premixed in one syringe of a stopped-flow spectrophometer and rapidly mixed
with O2 in a second syringe. Oxyferrous cyt P450 forms within 20 msec and is subsequently rapidly reduced by the
FMNH2 of the reductase. Cyt P450 returns to the Fe+3 state
with first order rate constants of 0.09 and 0.012s-1. Interestingly, the reductase oxidizes more quickly than cyt P450.
In a parallel experiment with Fe+2 cyt b5 the oxyferrous cyt
P450 turned over and returned to the Fe+3 state with rate
constants of 6.5 and 0.1s-1. When benzphetamine was the
substrate, the reaction was 53% coupled in the presence of
cyt b5 and 33% coupled with the reductase.
TP24
HUMAN HEPATIC OXIDATION
OF DOTHIEPIN MEDIATED BY
CYP2D6
HEQUN YIN, PHI TRAN, and VOLKER FISCHER
Department of Preclinical Safety, Novartis Biomedical Research Insistitute, One Health Plaza, East Hanover, NJ
07936, USA, [email protected]
Dothiepin, 3-(6H-dibenzo[b,e]thiepin-11-ylidene)propyldimethyl amine, is a tricyclic antidepressant, which is
composed of predominantly the E-isomer (95%) and a minor amount of the Z-isomer (5%). Human in vivo metabolism of dothiepin was reported to involve the formation of
nordothiepin, S-oxide, nordothiepin-S-oxide, and a N-glucuronide. The current study was undertaken to 1) further investigate the oxidative metabolism of dothiepin in vitro, and
2) to characterize the human hepatic CYP isoforms involved in the metabolism and the associated kinetic characteristics. Pooled human liver microsomes as well as individual cDNA-expressed human CYP isoforms were incubated
with dothiepin at various concentration levels. The metabolites formed were quantitated by HPLC with UV detection,
and structurally characterized by mass spectrometry and
comparison to synthetic standards. The kinetic parameters
were estimated using the Michaelis-Menten equation. Microsomal incubations produced an S-oxide and an N-desmethyl metabolite as the major products (Fig. 1). Minor products included an N-oxide, which is a newly identified metabolite, a monohydroxylation product, and a double N-demethylation product. Kinetic analysis indicated that the Soxidation involved a high affinity component with a Km of
1.7 µM and CLint of 26 µL/mg/min, and a low affinity component with a Km of 100 µM and CLint of 1.9 µL/mg/min.
Screening with cDNA-expressed CYP isoforms indicated
the involvement of CYPs 2D6, 3A4, and 1A2 in dothiepin
metabolism. Kinetic analysis indicated CYP2D6 as the high
affinity component with a Km of 2.0 µM and CLint of
3.2 µL/pmol/min, and CYPs 3A4 and 1A2 likely the low
affinity components with Km of 192 and 68 µM, and CLint
of 0.03 and 0.003 µL/pmol/min, respectively. The study
suggests that CYP2D6 is likely the major isoform responsible for the hepatic S-oxidation of dothiepin in human.
TP25
SELECTIVE DEHYDROGENATED INTERMEDIATES ARE
MECHANISM-BASED
INACTIVATORS OF CYP3A4,
CYP2E1, AND CYP2F1
KONSTANTINE W. SKORDOS, STACY J. SMEAL,
CHRISTOPHER A. REILLY, DIANE L. LANZA, and
GAROLD S. YOST
of Utah, 30 South 2000 East, Room 201, Salt Lake City, UT
84112, USA. email: [email protected]
P450-Mediated dehydrogenation of many substrates leads to the production of electrophilic intermediates, such as
quinones, quinone methides, and imines. Some of these
electrophiles are long-lived enough to be released from the
catalytic site and initiate toxicity. Examples are acetaminophen, equine estrogens, troglitazone, 3-methylindole,
valproic acid, and N-methylformamide. Conversely, several of the dehydrogenated intermediates are reactive
enough to alkylate active-site nucleophilic residues of the
P450 proteins, producing inactive enzymes (phencyclidine
Fig. 1.
S127
and the dehydrogenated electrophiles are potent mechanism-based inactivators of the unsuspecting enzymes. This
research was supported by USPHS grants (HL60143 and
HL13645) and by the National Institute of Standards and
Technology (Contract# 60NANBOD0006).
Fig. 1.
TP26
- CYP2B6, furafylline - CYP1A2, hydroxytamoxifen CYP2B6). We have initiated an examination of the catalytic mechanisms that control enzyme selectivity in the production of dehydrogenated intermediates, and the propensity of these electrophiles to produce mechanism-based inactivation. Substrates and enzymes used for these studies
were zafirlukast (CYP3A4), capsaicin (CYP2E1), and 3methylindole (CYP2F1), see Figure 1. These substrates
were oxidized by their respective enzymes to dehydrogenated imines which were characterized by trapping the
electrophiles with N-acetylcysteine or glutathione, followed by mass spectral and NMR structural identification. In
all three cases the dehydrogenated imines were major products, and were selectively produced by the enzyme, e.g.
the zafirlukast dehydrogenated intermediate was not produced by CYP2E1 and the 3-methylindole dehydrogenated
intermediate was not made by CYP3A4 or CYP2E1. In addition to catalyzing the selective formation of the dehydrogenated products, each enzyme was inactivated in a timeand concentration-dependent manner by its respective substrate. Again, selectivity was observed for the mechanismbased inactivation events, i.e. CYP3A4 was not inactivated
by capsaicin or 3-methylindole, and CYP2E1 was not inactivated by zafirlukast or 3-methylindole. Thus, these studies demonstrated that the selective dehydrogenation of
these diverse chemicals is catalyzed by certain enzymes,
EVA ANZENBACHEROVÁa, JANA BARANOVÁb,c,
ROMAN ZUBERa,b, PAVEL ANZENBACHERb,
PAVEL SOUâEKd
S128
HUMAN AND MINIPIG
CYTOCHROME P450 2E1
a
Institute of Medical Chemistry and Biochemistry and
Institute of Pharmacology, Faculty of Medicine, Palacky
University, Hnûvotínská 3, CZ-775 15 Olomouc,Czech
Republic and cSlovak Technical University, Bratislava,
Slovakia ,dStátní zdravotní ústav, Centrum HPNP, ·robárova 48, 100 42 Praha, Czech Republic
[email protected]
b
The search for a good model of drug metabolism in man
has led to intensive studies on CYP systems in other species1. The pig (or minipig) liver microsomal fraction as well
as the partially purified proteins has been shown to exhibit
enzyme activities characteristic of human CYP enzymes2.
The minipig cytochrome P450 form 2E1 has been recently purified to homogeneity and its primary structure
has been obtained3. A detailed comparison of the properties
of this enzyme with those of the human CYP2E1 is presented here. The minipig protein is well recognized by the
antibodies against human CYP2E1. This contribution also
shows the results of determination of both the prototypic
CYP2E1 activities i.e. the chlorzoxazone 6-hydroxylating
and the p-nitrophenol hydroxylating properties determined
in the microsomal fractions of minipig and human origin as
well as in the respective reconstituted systems. Inhibition
studies with diethyldithiocarbamate complement the study.
Both the chlorzoxazone 6-hydroxylating activity and
the p-nitrophenol hydroxylating one has been determined
using the methods described in literature4,5. The protein
itself has been isolated according to procedure published
earlier2,3. Enzyme kinetics has been analyzed using a LSW
Data Analysis software (www.lsw.com). The results obtained show conclusively that the minipig cytochrome P450
2E1 is able to catalyze both reactions exhibiting the activity well comparable to this of the human enzyme. Diethyldithiocarbamate, a specific CYP2E1 inhibitor, has
been also shown to be functional in minipig reconstituted
system inhibiting its activity to 10% and less. Hence, the
minipig enzyme shows the characteristics typical of
a CYP2E1 enzyme.
in the region of the C-helix for members of the CYP4 family. In order to test the significance of this finding, we
used the sequence of CYP4A1 in the conserved region to
search DNA databases. The conserved sequence detected
exclusively members of the CYP4 family, and two additional partial EST sequences.
Subsequent cloning of the two novel gene sequences
was achieved by RACE-PCR, and yielded full-length sequences for CYP4Z1 and CYP4X1. These data are consistent with subsequent human genomic sequences. The sequence of CYP4Z1 and CYP4X1 predict functional cytochromes P450. Antisera were raised against recombinant
fragments of CYP4X1 and CYP4Z1. These antisera are
characterised. The tissue-specific distribution of CYP4Z1
and CYP4X1 is discussed.
Acknowledgment
The authors thank the Grant Agency of the Czech Republic for support through the 203/02/1152 project.
1. Gibson G.G., Orton T.C., Tamburini P.P.: Biochem. J.
203, 161 (1982).
2. Hardwick J.P., et al.: Biol. Chem. 1987. 262, 801.
3. Christmas P., et al.: J. of Biol. Chem. 2001. 276, 38166.
4. Fisher M.B., Zheng Y.M., Rettie A.E: Biochem. Biophys. Res. Commun. 248, 352 (1998).
5. Kikuta Y., et al.: FEBS Lett. 348, 70 (1994).
REFERENCES
1. Zuber R., Anzenbacherová E., Anzenbacher P.: J. Cell.
Mol. Med. 6, 189 (2002).
2. Soucek P., Zuber R., Anzenbacherová E., Anzenbacher
P., Guengerich F.P.: BMC Pharmacology 1, 11 (2001).
3. Anzenbacherová E., Anzenbacher P., Zuber R., Souãek
P.: Drug Metab. Revs. 2003, in print (8th Eur. ISSX Meeting Proceedings)
4. Lucas D., Berthou F., Girre C., Poitrenaud F., Ménez
J.F.: J. Chromatogr. B 622, 79 (1993)
5. Tassaneeyakul W., Veronese M., Birkett D.J., Miners
J.O.: J. Chromatogr. B 616, 73 (1993)
REFERENCES
TP28
POLYMORPHISM OF P450 IN
DROSOPHILA MELANOGASTER
ALEXANDRA BRUN-BARALE, DIDIER CROCHARD,
SOPHIE TARES, LAURY ARTHAUD,
JEAN-MARC BRIDE, and MARCEL AMICHOT
INRA, UMR Rose 1112 API, 123 boulevard Francis Meilland, 06606 Antibes cedex, [email protected]
TP27
TWO NOVEL HUMAN P450
ENZYMES, CYP4Z1 AND CYP4X1
NEILL J HORLEY, TAO JIANG, and DAVID R BELL
School of Life and Environmental Sciences, University of
Nottingham, University Park, Nottingham, NG7 2RD. UK
The CYP4 family was originally discovered as the lauric acid omega-hydroxylase, which was induced in rat liver
by peroxisome proliferators1,2. The cloning of members of
the CYP4B and CYP4F families has revealed that there are
additionally fatty acid substrates for the CYP4 family 2-5.
The CYP4 family has a potentially important role in metabolism of endogenous fatty compounds.
We prepared an extensive alignment of members of the
CYP4 family using the program SAGA; this alignment included both vertebrate and invertebrate P450 proteins. The
alignment showed unexpectedly high sequence similarity
S129
Because of its impact on drug metabolism, cytochrome
P450s polymorphism is under the scope in humans1. In insects, they are involved in numerous instances of insecticide resistance and adaptation to chemical stresses. Available large collections of strains, easyness of wild populations collections and genome knowledge make Drosophila
melanogaster a suitable species for a global study of
P450s polymorphism in insects. In this first study, we analyzed in 7 strains the polymorphism of Cyp6a2 and Cyp6g1
(xenobiotics metabolism and insecticide resistance2,3),
Cyp302a1 and Cyp315a1 (steroid synthesis, dib and sad
genes4,5) and of Cyp6w1 and Cyp6u1, two P450s physically
close to Cyp6a26.
For each gene, primers were designed to amplify by
PCR fragments no longer than 320 bp in the coding sequence. We first analysed these fragments by SSCP. Results
showed that Cyp6a2, Cyp6g1 and Cyp302a1 (dib) were polymorphic with 6, 4 and 5 alleles respectively contrary to
Cyp315a1 (sad) which had only 2 alleles. Work is in
TP29
BACTERIAL MONOOXYGENASES IN BIOTRANSFORMATION
AND BIODEGRADATION OF
ALIPHATIC AND AROMATIC
COMPOUNDS
VICTOR LEONT’EV, TAT’ANA ACHRAMOVITCH,
IRINA BURAK, and OLGA IGNATOVETS
Belorussian State Technological University, Sverdlova 13a, Minsk, 220050 Belarus, [email protected]
Fig. 1. The 4 genes were represented wtih the position and the size
of the introns. The polymorphic regions are indicated under the
gene with black bars and narrow bars respectively.
progress with Cyp6u1 and Cyp6w1. There was a region polymorphic in all the genes which code for the G, H and
I helices but polymorphism was not distributed among the
genes uniformously. Although polymorphism was expected for Cyp6a2 and to a lesser extend for Cyp6g1, we were
surprised to classify Cyp302a1 in the polymorphic
P450s category. Indeed, this gene is involved in steroid
hormone synthesis and thus should be submitted to a high
selection pressure. Nevertheless, we are sequencing these
alleles to identify which polymorphism event is translated
and thus putatively effective on protein function.
The amount of polymorphism of each gene, its repartition among the gene and its putative effects on the encoded
protein activity will be discussed taking account of the
function of the gene and P450 structure/activity relationships.
REFERENCES
1. http://www.imm.ki.se/CYPalleles
2. Berge J.B., et al. : Philos. Trans. R. Soc. London 353,
1701 (1998).
3. Daborn P.J., et al. : Science 297, 2253 (2002).
4. Warren J.T., et al. : PNAS 99, 11043 (2002).
5. Marcela Chavez V., et al. : Development 127, 4115
(2000).
6. http://p450.antibes.inra.fr
S130
Bacteria are able to degrade or transform many natural
or xenobiotic compounds occurring in the environment
using variety of oxidative reactions. An important group of
enzymes are the monooxygenases including P-450 enzymes. The best known and characterized bacterial P-450
was the P-450cam isolated from Pseudomonas1.
Study of dependent of the levels of cytochromes b5 and
P450 from structure of substrate was by purpose of this work.
Two series of the compounds were used for these investigations. Aliphatic and alicyclic compounds represent the first,
halogenated aromatic compounds represent the second series.
Pseudomonas fluorescens B-22 was used for the transformation aliphatic and alicyclic compounds, Rhodococcus opacus
B-2243 for the degradation of halogenated aromatic compounds. The levels of cytochromes b5 and P450 was determined spectrophotometrically by the method Omura and Sato2.
The concentrations of cyt P450 were calculated from reduced
carbon monooxide difference spectrum, using an exinction
coefficient 92,8 mM-1 cm-1 in according to the paper3. The results of these studies are represented in Table 1 and Table 2.
The change of the levels of cytochromes b5 and P450 in
Pseudomonas fluorescens B-22 (Table 1) probably is caused by distinction of chemical reactions on cyt P450. Hexan is capable to hydroxylate with formation of alcohols;
hexen-1 may be hydroxylated, but preferable it epoxydates; cyclohexen and nonen-4 epoxydate, what was confirmed in further study with GC.
Table 1. Content of cytochromes b5 and P450 in Pseudomonas fluorescens B-22
Substrate
Contents
of cytochrome b5,
nmol/mg of protein
Contents
of cytochrome P450,
nmol/mg of protein
Exponential
growth
phase
Exponential Stationary
growth
growth
phase
phase
Stationary
growth
phase
Glucose
0,04
0,02
0,02
-
Hexan
0,12
0,05
0,10
0,08
Hexen-1
0,18
0,03
0,21
0,03
Cyclohexen
0,04
0,04
0,23
0,02
Nonen-4
0,05
0,01
0,18
0,02
Table 2. Content of cytochromes b5 and P450 in Rhodococcus
opacus B-2243
Substrate
Contents
of cytochrome b5,
nmol/mg of protein
Contents
of cytochrome P450,
nmol/mg of protein
Exponential
growth
phase
Exponential Stationary
growth
growth
phase
phase
Stationary
growth
phase
Benzen
0,010
0,003
0,033
0,012
Chlorbenzen
0,016
-
0,077
-
Bromobenzen
0,020
0,009
0,040
0,016
This presentation will focus on the use of rabbit as an
animal model to predict CYP3A-drug drug interactions.
First, in vitro studies were conducted to show that the metabolism of known CYP3A substrates (testosterone, DFP1,
DFB2 (Figure 1)) and a variety of chemical inhibitors behaved similarly in human and rabbit. In vivo studies were
then conducted and showed that DFB, being a fast clearance drug in rabbit, would be suitable to evaluate the effect of CYP3A inhibitors while DFP, a slower clearance
drug, had the proper pharmacokinetic behaviors to test the
effect of an inducer. When ketoconazole was co-administered with DFB to rabbit, the AUC of DFB increased by
4-fold. When rabbits were treated with rifamipicin, the
All compounds used in the experiments with Rhodococcus opacus B-2243 were the growth substrates. From our
point of view, the change of the levels of cytochromes b5
and P450 in Rhodococcus opacus B-2243 may be explained by next causes:
1) in the process of degradation of the growth substrates cyt P450 may be catalyzes the different stages of transformation of these compounds;
2) cyt P450 may be catalyzes the same stage of transformation of these compounds, but P450 has different substrate specificity to those compounds, that are substrate for
it in this reaction.
REFERENCES
1. Hedegaard J., Gunsalus I.C.: J. Biol. Chem. 240, 4038
(1965).
2. Omura T., Sato R.: Biol. Chem. 239, 2370 (1964).
3. Gunsalus I. C., Wagner G. C.: Meth. Enzymol. 57, 166
(1978).
TP30
RABBIT AS AN ANIMAL MODEL
TO STUDY CYP3A-MEDIATED
DRUG-DRUG INTERACTIONS
NATHALIE CHAURET, LEKHA SLENO, JOSÉ SILVA,
ROBERT HOULE, STEPHEN DAY, and DEBORAH NICOLL-GRIFFITH.
Merck Frosst Centre for Therapeutic Research, P.O. Box
1005, Pointe-Claire-Dorval, Québec, Canada, H9R 4P8.
[email protected]
Cytochrome P450 3A (CYP3A) is an important class of
enzymes highly involved in the metabolism of drugs. Any
new drugs that can alter CYP3A levels through induction
or inhibition can eventually cause drug-drug interactions in
humans. There are several in vitro assays that have been
developed to look at the inhibition or induction of
CYP3A using human liver subcellular fractions or hepatocytes. The in vivo relevance of the results obtained from
these assays is, however, difficult to address.
S131
Fig. 1. Structure of DFP and DFB
AUC of DFP decreased by 50%, consistent with the effect
of a CYP3A inducer. This CYP3A induction was also confirmed by Western blot analysis of rabbit livers. This model has also been used in our drug discovery program to
test if new chemical entities can be victims of CYP3A interactions. In these studies, the effect of ketoconazole or rifampicin on the clearance of the NCE entities is monitored.
This poster will provide the details of the in vitro and in
vivo profiles of DFB and DFH in rabbits and will describe
the rabbit protocol used to evaluate whether a NCE is likely to be victim of CYP3A interactions in humans.
REFERENCES
1 Nicoll-Griffith D.A.et al.: Drug Metabol. Disp. 29, 159
(2000)
2 Chauret N. et al.: Anal. Biochemistry, 276, 215(1999)
TP31
The groups of patients with and withouth EPS did not differ regarding CYP2D6 genotype frequencies, predicted
phenotype or the number of functional CYP2D6 alleles, or
regarding the type and dosage of antipsychotics. PMs however scored significantly higher on the negative subscale
for PANSS (p = 0.008). Our results indicate that CYP2D6
polymorphism is not associated with the incidence of antipsychotic-induced EPS in patients with stabile remission
on long term maintenance treatment.
CYP2D6 POLYMORPHISM AND
EXTRAPYRAMIDAL SYMPTOMS
IN PATIENTS ON LONG TERM
ANTIPSYCHOTIC TREATMENT
VITA DOLÎANa, BLANKA KORES-PLESNIâARb,
BOJAN ZALARc, and KATJA BRESKVARa
a
Institute of Biochemistry, Faculty of Medicine, Ljubljana,
Slovenia, bDept. of Psychiatry, Teaching Hospital Maribor,
Maribor, Slovenia, cUniversity Psychiatry Clinic, Ljubljana,
Slovenia; [email protected]
Cytochrome P450 enzyme CYP2D6 is one of the most
important enzymes involved in metabolism of antipsychotic drugs. Due to genetic polymorphism of this enzyme coded by polymorphic CYP2D6 gene individuals differ in
drug metabolising capacity. CYP2D6 genotyping for the
most common defective alleles was reported to be a reliable prediction of decreased metabolic capacity for
CYP2D6 substrates1. Our aim was to asses the predictive
value of low and high resolution CYP2D6 genotyping for
the development of extrapyramidal symptoms in patients in
stable remission receiving long term maintenance antipsychotic therapy. 161 out-patients meeting the DSMIV criteria for schizophrenia receiving maintenance antipsychotic
therapy with either zuclopenthixole, haloperidol or risperidone were included in the study. None of them was receiving any concomittant medication that would have potentially inhibitory effect on CYP2D6. All were in stable remission and on present medication for 44.7 ± 37.5 months.
Extrapyramidal symptoms (EPS) were evaluated in 131 patients with Simpson-Angus rating scale (parkinsonism),
Barnes akathisia rating scale, and Abnormal Involuntary
Movement Scale (AIMS- tardive dystonia and tardive dyskinesia). Psychopathological symptoms were evaluated
with Positive and Negative Syndrome Scale for Schizophrenia (PANSS). CYP2D6 gene deletions (CYP2D6*5) and
duplications were identified by long-PCRs2,3. Next,
CYP2D6 genotyping of *1, *3, and *4 (low resolution) and
additionally of *2, *6, *8, *9, *10, *11, *12, *14 and *15
allele (high resolution) was performed by nested PCRRFLP approach after amplification of the entire CYP2D6
gene4. The frequency of CYP2D6*1 allele coding for extensive metabolizer phenotype (EM) was 0.427. The frequencies of alleles coding for slightly (*2) or moderately (*9,
*10) reduced activity (intermediate metabolizer phenotype- IM) were: 0.242, 0.016 and 0.041. The frequencies of
defective alleles (poor metabolizer phenotype- PM) were:
0.178 (*4), 0.035 (*3), 0.022 (*5 and *6), while *8, *11,
*12, *14 and *15 allele were not found. CYP2D6 duplication alleles were found with frequencies of 0.007 (*1X2
and *2X2) and 0.003 (*4X2). The high resolution genotyping did not reveal any additional PMs, but helped to identify IMs. Regarding the EPS parkinsonism was observed in
56/131 (42.8%) and akathisia in 15/131 (11.5%) patients.
S132
REFERENCES
1. Griese U.E., Zanger U.M., Brudermanns U. et al. Pharmacogenetics 8, 15 (1998).
2. Wennerholm A., Johansson I., Massele A.Y. et al. Pharmacogenetics 5, 215 (1995).
3. Lundquist E., Johansson I., Ingelman-Sundberg M.
Gene 226, 327 (1999).
4. Sachse C., Brockmöller J., Bauer S., Roots I. Am. J.
Hum. Genet. 60, 284 (1997).
TP32
CYP2U1, A THYMUS AND
CEREBELLUM EXPRESSED
CYTOCHROME P450 CONVERTS
ARACHIDONIC ACID INTO
BIOACTIVE DERIVATIVES
19- AND 20-HETE
CHRISTIAN HELVIG, SAM CHUANG,
MOHAMMED TAIMI, GLENVILLE JONES,
MARTIN PETKOVICH, JAY A. WHITE,
and BOZENA KORCZAK.
Cytochroma Inc., 330 Cochrane Drive, Markham,
ON L3R 8E4 CANADA.
The cytochrome P450 pathway for arachidonic acid
(AA) bioactivation plays an important functional role in
cell and organ physiology1. Arachidonic acid metabolites
generated by cytochrome P450s (CYPs) have been described as powerful bioactive mediators in vascular and renal
physiology and in the pathophysiology of experimental hypertension. However the full range of AA activities is not
yet clear. Characterization of cytochrome P450s involved
in AA metabolism is a critical step in this process.
We have recently cloned the cDNA encoding CYP2U1,
a previously uncharacterized member of the CYP2 family.
This protein shares 35, 34 and 31% amino acid identity
with CYP2D6, CYP2R1 and CYP2J2 respectively. The tissue distribution of this CYP was investigated by northernblot and by real-time PCR. Interestingly, we found that
CYP2U1 is predominantly expressed in the cerebellum and
thymus. CYP2U1 catalytic activity was tested in microsomal fractions from sf9 insect cells expressing CYP2U1 re-
Tab. 1. CYP2U1 substrates
Exogenous
Endogenous
Linolenic acid (C18:3)
Palmitic acid (C16:0)
Arachidonic acid (C20:4)
Palmitoleic acid (C16:1)
Eicosapentaenoic acid (C20:5)
Stearic acid (C18:0)
Eicosatrienoic acid (C20:3)
Vaccenic acid (C18:1)
Docosahexaenoic acid (C22:6) Eicosapentaenoic acid (C20:5)
Docosatetraenoic acid (C22:4)
Docosahexaenoic acid (C22:6)
combinant protein in the presence or absence of exogenous
substrate. Medium and long chain fatty acids (C16 to C22)
were metabolized by CYP2U1 (Table 1). In the case of AA,
the two metabolites were identified as the 20- and 19-hydroxyeicosatetraenoic acid (20- and 19-HETE). Both ketoconazole and 17-ODYA inhibit CYP2U1 catalytic activity.
Based on CYP2U1 amino acid sequence similarity and its
ability to generate bioactive eicosanoid derivatives, we postulate that CYP2U1 plays an important physiological role
both in the brain and thymus.
This is the first report of the cloning and functional expression of a new member of family 2, CYP2U1, which
metabolize medium and long chain fatty acids.
TP34
REFERENCES
1. Capdevila JH, Falck JR: Biochem Biopys Res Commun
2001, 285, 571.
TP33
led 51 and 41% identity with human CYP26B and
CYP26A, respectively. The genomic structure (intron-exon
organization) of CYP26C is identical to that of
CYP26B and differs from CYP26A by the number and the
position of introns. The CYP26C gene is localized on chromosome 10 (chromosome 19 in mouse) in close proximity
to the gene locus for CYP26A.
We have expressed recombinant CYP26C in insect
cells and determined its catalytic activity in the presence of
various retinoid substrates. All trans-retinoic acid (atRA)
was metabolized by CYP26C in a fashion similar to that by
CYP26A and CYP26B. However, CYP26C was also
shown to metabolize 9-cis-retinoic acid (9cRa) to the corresponding 4-oxo- and 4-OH-retinoic acid metabolites.
This property seems to be unique to CYP26C since other
retinoic acid metabolizing enzymes, CYP26A and
CYP26B do not recognized 9cRA as a substrate. Similarly,
although ketoconazole inhibits the activities of both
CYP26A and CYP26B, CYP26C activity is unaffected,
further evidence of differences in substrate binding properties amongst CYP26 family members.
We speculate that based upon the unique expression
pattern during embryonic development and substrate binding that CYP26C plays a role distinct from those of
CYP26A and CYP26B.
CDNA CLONING AND BIOCHEMICAL CHARACTERIZATION
OF CYP4F11, A NOVEL HUMAN
CYTOCHROME P450
CHRISTIAN HELVIG and BOZENA KORCZAK
A NOVEL CYP26 FAMILY
MEMBER METABOLIZES
9-CIS-RETINOIC ACID
ON L3R 8E4 CANADA.
MOHAMMED TAIMI, CHRISTIAN HELVIG,
MARTIN PETKOVICH, and BOZENA KORCZAK.
ON L3R 8E4 CANADA
Retinoids (vitamin A analogs) are potent regulators of
growth and differentiation of normal, pre-malignant and
malignant cells. The synthesis and breakdown of retinoic
acid are critical mechanisms governing the distribution
and responsiveness of retinoic acid in target tissues both
in the embryo and the adult. Retinoic acid catabolism is
mediated by a highly specific cytochrome P450 enzyme
family, called CYP26. Here we report the cloning and biochemical characterization of a new family member CYP26C.
A novel cytochrome P450 was cloned using human adrenal poly(A) RNA. CYP26C amino acid sequence revea-
S133
The CYP4F subfamily is known to be involved in the
hydroxylation of arachidonic acid (AA), lipoxin, tocopherol (vitamin E), leukotriene B4 (LTB4), prostaglandins and
ebastine (antihistamine). Members of the CYP4F subfamily are also known to play an important functional role in
inflammatory responses by regulation of the level of inflammatory mediators and in the biotransformation of certain xenobiotics.
Here we report the biochemical properties of a new
member of the CYP4F subfamily, CYP4F11. The CYP4F11
amino acid sequence has 82, 80, and 79% identity with other members of the CYP4F subfamily CYP4F3, CYP4F2
and CYP4F8 amino acid sequences, respectively 1.
CYP4F11 catalytic activity was tested in microsomal
fractions from sf9 insect cells expressing CYP4F11 recombinant protein in presence or absence of exogenous substrate. Our data show that arachidonic acid (AA), leukotriene B4 (LTB4) and other short to long chain (C12 to C22)
Tab. 1. CYP4F11 substrates:
Exogenous
Endogenous
LTB4
Lauric acid (C12:0)
Myristic acid (C14:0)
Palmitic acid (C16:0)
Palmitoleic acid (C16:1)
Stearic acid (C18:0)
Vaccenic acid (C18:1)
Docosahexaenoic acid (C22:6)
saturated or unsaturated fatty acids were metabolized
by CYP4F11 (Table 1). In the case of AA, the unique metabolite formed was identified by mass-spectrometry as
20-hydroxyeicosatetraenoic acid (20-HETE). An apparent
Km and Vmax of 41 µM and of 2 nmol/min/mg of protein
were measured for AA. Based on CYP4F11 catalytic activity we can postulate that CYP4F11 might play an important functional role in inflammatory response. In specific
tissues, it might also be involve in the formation of the
physiologically active 20-HETE.
This is the first report of the functional characterization
of a new member of the subfamily 4F, CYP4F11, which
metabolize fatty acids and leukotriene B4.
REFERENCES
1. Cui X, Nelson DR, Strobel HW: Genomics 68, 161
(2000).
TP35
CYP2C9 POLYMORPHISM
AND WARFARIN DOSE
REQUIREMENT
DARJA HERMANa, TINA GLOBOKARb,
POLONA PETERNELb, MOJCA STEGNARb,
KATJA BRESKVARa, and VITA DOLÎANa
a
Institute of Biochemistry, Faculty of Medicine, Ljubljana,
Slovenia, bUniversity Medical Centre, Trnovo Hospital of
Internal Medicine, Department for Vascular Diseases,
Ljubljana, Slovenia, [email protected]
Warfarin is the most widely prescribed oral anticoagulant drug in Slovenia. Its narrow therapeutic index and high
interindividual variability in the dose required to achieve
the desired therapeutic effect complicate the treatment.
Cytochrome P450 2C9 (CYP2C9) is largely responsible for
terminating the anticoagulant effect of racemic warfarin
via hydroxylation of the pharmacologically more potent
S-enantiomer to inactive metabolites. Genetic polymorphisms of the CYP2C9 gene, which give rise to proteins
S134
with decreased catabolic activity, have been described. The
variant alleles include CYP2C9*2 with a point mutation in
exon 3 causing an Arg144Cys exchange, and CYP2C9*3
with a point mutation in exon 7 resulting in an Ile359Leu
exchange1.
The aim of this study was to asses the influence of
CYP2C9 polymorphism on the dose requirement of the anticoagulant drug warfarin.
In total 157 patients receiving warfarin from 6 months
to 14 years (average 3,5 years) were included in the study.
Indication for warfarin treatment were atrial fibrillation
(n=145) and mechanical heart valves (n=12). The patients
were divided into three groups according to the dose requirement: low (LD: < 2,15 mg/day, n=38), medium (MD:
2,5-7 mg/day, n=103) and high (HD: > 7 mg/day, n= 16).
CYP2C9 polymorphisms were analysed by PCR amplification of genomic DNA followed by restriction enzyme analysis as previously described by Yasar et al.2
In the LD group 13 patients (34.2%) were homozygous
for wild-type allele (*1/*1), 20 patients (52.6%) were heterozygous and 5 patients (13.2%) were homozygous for polymorphic alleles. In the MD group 74 patients (71.8%)
had *1/*1 genotype, 27 patients (26.2%) were heterozygous and 2 patients (2.0%) were homozygous for polymorphic alleles. In the HD group all the patients were homozygous for wild-type allele. The frequencies of
CYP2C9*3 polymorphic alleles were significantly higher
in LD as compared to MD group (26.3% as compared to
4.9%). The average dose of warfarin was the highest
among patients with *1/*1 genotype (4.42 mg/day) and
was decreasing with the number of polymorphic alleles:
2.96 mg/day in *1/*2, 2.71 mg/day in *1/*3, 2.14 mg/day
in *2/*3 and 1.00 mg/day in *3/*3 genotype. The only exception was the patient who had *2/*2 genotype and the
warfarin dose 4.29 mg/day.
CYP2C9 genetic polymorphisms significantly contribute to individual warfarin dose requirement. Since
CYP2C9 polymorphisms are common in Slovenian population, occurring in 34% of individuals3, it is of potential
clinical importance to be able to identify patients carrying
polymorphic alleles when aiming for rational and individualised anticoagulant therapy with warfarin.
REFERENCES
1. Lee C.R., Goldstein J.A., Pieper J.A.: Pharmacogenetics. 12, 251 (2002).
2. Yasar U., Eliasson E., Dahl M.L., Johansson I., Ingelman-Sundberg M., Sjoqvist F.: Biochem. Biophys Res
Commun. 254, 628 (1999).
3. Herman D., DolÏan V., Breskvar K.: 2nd international
FEBS advanced course: Cytochrome P450 systems:
from structure to application, Kranjska gora, 21-26 May
2002, Book of Abstracts (Cresnar B., DolÏan V., Rozman D., eds.), p.110.
TP36
A STREPTOMYCES CYP105D1
EXPORTED IN ESCHERICHIA
COLI INDUCES PERIPLASMIC
ACCUMULATION OF
PORPHYRIN
TP37
NOVEL SNP’S IN THE CYP2B6
GENE AND THEIR FUNCTIONAL
CONSEQUENCES
KATHRIN KLEINa, THOMAS LANGb,
TANJA RICHTERa, MICHEL EICHELBAUMa,
MATTHIAS SCHWABa, and
ULRICH M. ZANGERa
M. KALIM AKHTAR, NAHEED N. KADERBHAI, and
MUSTAK A. KADERBHAI
Institute of Biological Sciences, Cledwyn Building, University of Wales, Aberystwyth, Ceredigion, Wales, SY23 3DD,
United Kingdom, [email protected]
Porphyrias are clinical conditions arising from genetic
or sporadic defects of the enzymes in haem biosynthesis1.
These diseases are associated with the accumulation of oxidised haem intermediates known as porphyrins. The commonest porphyric disorder, porphyria cutanea tarda, is
characterised by hepatic accumulation and excretion of
uroporphyrins (I and III). A number of etiological factors
are thought to precipitate the sporadic form of the disease
including iron overload, ascorbic acid deficiency, hepatitis
c infection and induction of P450 isoenzymes. A structurally competent Streptomyces griseus, CYP105D1 was expressed in aerobically-grown Escherichia coli as an exportable form2. Expression of CYP105D1 was time-dependently coupled with accumulation of a stable, highly fluorescent compound in the periplasmic space, which was isolated and identified as uroporphyrin. The accumulation of
uroporphyrin was over a hundred-fold higher than cells
lacking CYP105D1. Expression of a cytoplasmic-resident
CYP105D1 at a comparative level to the secreted form was
far less effective in promoting porphyrin accumulation in
the cytoplasm or periplasm. An E. coli strain that exported
a mammalian holo-cytochrome b5 at ten-fold molar excess
over the exported CYP105D1 produced uroporphyrin levels similar to the control strain. This negated the possibility that the oxidative production of uroporphrin in the
CYP105-secreted strain was a consequence of 5-aminolevulinic acid-promoted synthesis of heme precursor to provide the prosthetic group for the over-produced apo-cytochromes. Uroporphyrin production was abolished in the
CYP105D1 secretory strain by azole-based P450 inhibitors. Furthermore, the isolated secretory CYP105D1, when
reconstituted in vitro, was active in its ability to convert
uroporphyrinogen I to uroporphyrin I. The present study
demonstrates that the exported CYP105D1 catalyses periplasmic conversion of uropophyrinogen to uroporphrin in
E. coli.
a
b
Epidauros Biotechnology AG, Am Neuland 1, D-82347
Bernried, Germany; [email protected]
The human cytochrome P450 2B6 is involved in the metabolism and activation of several therapeutically drugs and
environmental toxins such as bupropion, cyclophosphamide, nicotine and aflatoxin B. The CYP2B6 gene is highly
polymorphic and at least 9 alleles with amino acid exchanges and associated with changes in liver protein expression
are presently known1, 2. By further sequencing of CYP2B6 in
samples from Caucasians we found five new point mutations (A-E) in coding regions. These novel SNPs are predicted to result in amino acid exchanges in exon1 (A: M46V),
exon2 (B: G99E), exon3 (C: K139E; D: R140Q) and exon8
(E: I391N). Comparison of hepatic CYP2B6 protein levels
and bupropion hydroxylase activity between polymorphic
(n=10) and wildtype (n=31) samples demonstrated significantly reduced CYP2B6 content (p=0.0118) and enzyme
activity (p=0.001). To investigate the functional concequences of the amino acid substitutions, enzyme variants
A-E were recombinantly expressed in insect cells using baculovirus expression system and COS1 cells. Analysis of
CO difference spectra of infected insect cells showed lower
P450 content but higher P420 amount for the variant proteins 2B6.A, 2B6.B, 2B6.C, and 2B6.E, whereas 2B6.D was
comparable to WT. Immunoblots of infected insect cells
showed nearly the same amount of 2B6 protein for 2B6.A,
2B6.B and 2B6.D compared to wildtype. In the case of
2B6.C and 2B6.E, almost no immunostainable protein was
detected. These results demonstrate that the novel 2B6 variants A, B, C and E are functionally severely compromized
and may lead to a poor metabolizer phenotype if present in
homozygous form. Further characterization of the new variants is currently in progress.
(grant ZA 245/2-1), and the Robert Bosch Foundation
Stuttgart.
REFERENCES
REFERENCES
1. Sassa, S. Kappas, A.: J Intern. Med. 247, 169 (2000).
2. Kaderbhai, M. A., Ugochukwu, C. C., Kelly, S. L.,
Lamb, D. C.: Appl. Environ. Microbiol. 67, 2136
(2001).
S135
Hofmann U, Eichelbaum M, Schwab M, Zanger UM.
Pharmacogenetics 11, 399 (2001).
TP38
COMPARATIVE ANALYSIS OF
CYP3A EXPRESSION IN HUMAN
LIVER SUGGESTS ONLY
A MINOR ROLE FOR CYP3A5
IN DRUG METABOLISM
TP39
SARAH MALMEBOa, ANNA WESTLIND-JOHNSSONa,
ANNA JOHANSSONb, CHARLOTTA OTTERb,
TOMMY B ANDERSSONc, INGER JOHANSSONa,
ROBERT J EDWARDSd, ALAN R BOOBISd, and
MAGNUS INGELMAN-SUNDBERGa
SEARCHING FOR THE GENE
ENCODING STEROID
11β-HYDROXYLASE OF THE
FILAMENTOUS FUNGUS
COCHLIOBOLUS LUNATUS
ENEJ KU·âERa, LJERKA LAHb,
RADOVAN KOMELAb
a
a
Institute of Environmental Medicine, Division of Molecular Toxicology, Karolinska Institutet, Stockholm, Sweden,
b
Molecular Biology, AstraZeneca Mölndal R&D, Mölndal,
Sweden; cDMPK & BAC, AstraZeneca Mölndal R&D,
Mölndal, Sweden; dSection of Clinical Pharmacology, Imperial College School of Medicine, London, UK
[email protected]
In order to study mechanisms behind the interindividual variability in CYP3A expression and the relative contribution of the different CYP3A enzymes to the overall
CYP3A activity we have analysed CYP3A4, CYP3A5,
CYP3A43 and PXR mRNA and CYP3A4 and CYP3A5
protein expression, catalytic activity and polymorphism in
the CYP3A5 gene in a panel of 46 Caucasian human livers.
Protein quantification was performed by Western blotting
using enzyme-specific antibodies directed to the C-termini
of CYP3A4 or CYP3A5, and carrier protein-coupled peptides as standards. The mRNA levels were determined by
quantitative real-time PCR. CYP3A activity was measured
by analysis of the rate of testosterone 6(-hydroxylation.
A correlation existed between all CYP3A and PXR mRNA
transcripts measured. The interindividual variability in
CYP3A4 and CYP3A5 mRNA expression was higher than
of CYP3A protein and activity. The CYP3A5 protein was
expressed at quantifiable levels in 5 (10.9%) of the livers.
Four of those were heterozygous for the CYP3A5*1 allele
and had CYP3A5 protein at a mean level of 17% of that of
total CYP3A, whereas one liver sample was from
a CYP3A5*3 homozygote individual having lower amounts
of CYP3A5. In total, CYP3A5 only contributed 2% of the
overall CYP3A protein among all samples. In conclusion,
our data indicate that CYP3A4, CYP3A5, CYP3A43 and
PXR hepatic mRNA expression correlate, indicating common regulatory features, and that the contribution of
CYP3A5 to hepatic drug metabolism in Caucasians is insignificant.
S136
National Center for Molecular Biology, Faculty of Medicine, Vrazov trg 2, SI-1000, bLjubljana, Slovenia, National
Institute of Chemistry, Hajdrihova 19, SI-1000, Ljubljana,
Slovenia
11β-hydroxylation of steroids is one of the key steps in
the production of corticosteroids. The enzymes responsible
for the hydroxylation of steroids by filamentous fungi belong to the cytochrome P450 superfamily. Despite the substantial biotechnological importance of these enzymes, the
sequences of the genes encoding fungal steroid 11β-hydroxylase, or any other fungal steroid hydroxylase, have not
yet been identified. The primary structures of these enzymes have also not yet been described. This is in part due to
the low specific content of the protein, difficulties associated with their isolation (membrane bound enzymes), the
presence of multiple cytochrome P450 isoenzymes, and the
lack of homology with the analogue enzymes of higher eukaryotes. The filamentous fungus Cochliobolus lunatus is
an important phytopathogen, capable of 11β-hydroxylation
of steroids. Based on the conserved, cytochrome P450 specific regions, and the nucleotide sequence of the benzoate
para-hydroxylase of the related filamentous fungus Aspergillus niger, a 273 bp long gene fragment has been obtained
using the PCR method. The fragment was sequenced, and
the BLAST database search clearly showed its cytochrome
P450 nature. However, it could not be determined, whether
the fragment belonged to the gene encoding steroid 11β-hydroxylase, or perhaps some other cytochrome P450 gene
which could be expected in this fungus. Based on the sequence of the obtained 273bp fragment, new cytochrome
P450 specific primers were designed. Using the rapid amplification of cDNA ends method, an entire 3’-end of the
gene, and a part of the 5’-end were successfully amplified
from the induced mycelium. After cloning, the sequence of
the 3’-end was determined using an automatic sequencer.
Using the BLAST database search, the obtained 3’-end was
found to have a high homology with numerous cytochromes
P450, including the benzoate para-hydroxylase of A. niger.
The obtained 3’-end will be used as a probe for screening
genomic and cDNA libraries. Thus the entire gene to which
the 3’-end belongs will probably be identified, as well as,
with a slightly lower hybridization intensity, other cytochrome P450 genes present in the fungus. It is likely that,
among these, the steroid 11β-hydroxylase would be found
as well.
REFERENCES
and function, including investigation of their interaction
with CYP partners are presented.
1. Hoerhold C., Undisz K., Groh H., Sahm R., Schade W.,
Komel R.: J.Basic. Microbiol., 26, 335 (1986).
2. Jaenig G.R., Pfeil D., Miller-Frohne M., Reimer H.,
Henning M., Schwarze W., Ruckpaul K.: J. Steroid Biochem. Mol. Biol., 43, 1117 (1992).
3. Komel R., Rozman D., Vitas M., Drobniã K., Kelly
S.L.: Cytochrome P450 8th International conference,
Paris, 1994. Lechner, M.C. (ed.) Paris, John Libbey Eurotext: 805 (1994).
4. Suzuki K., Sanga K., Chikakoka Y., Itagaki E.:Biochim.
Biophys. Acta, 1203, 215 (1993,).
5. Van Gorcom R.F.M., Boschloo J.G., Kuijvenhoven A.,
Lange J., Van Vark A.J., Bos C.J., Van Balken A.M.,
Pouwels P.H., Van den Hondel C.A.M.J.J.: Mol. Gen.
Genet., 223, 192 (1990).
6. Vitas M., Pajic T., Kellz S.L., Kome. R.: J. Steroid. Biochem. Molec. Biol., 63, 345 (1997).
TP40
TRANSCRIPT ANALYSIS
AND AZOLE INHIBITION OF
MYCOBACTERIAL CYPS
JOSIE PARKER, COLIN JACKSON, TIM MARCZYLO,
SAMIRAH PERALLY, DAVID LAMB, STEVEN KELLY
and DIANE KELLY.
Wolfson Laboratory of P450 Biodiversity, Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, Wales, UK. [email protected]
Mycobacteria contain novel CYPs of unknown function
and revealing their roles and potential application is a challenge for the coming years. One potential application is in
the treatment of mycobacterial infection. Known CYP inhibitors among the antifungal drugs have been observed to
show effect against various fast-growing bacteria1, but reduced effect against M. bovis BCG2. The presence of
a CYP51 in mycobacteria is the most obvious potential target of such compounds as in fungi, but azole efficacy could
easily be due to effect on other CYPs in these organisms.
We have looked at the CYP51 locus in mycobacteria
and observed it is conserved except in M. leprae. CYP513
and the surrounding genes appear to be part of an operon.
All the ORFs in the operon have been shown to be transcribed in growing M. smegmatis and M. bovis BCG. We are
also examining the CYP51 azole affinity in M. tuberculosis and of M. smegmatis and have observed a correlation to
the anti-mycobacterial activity of the compounds.
Other CYPs bind azole compounds including
CYP162A2 the M. smegmatis homologue of the lone
M. leprae CYP, but in some cases novel features are encountered as with CYP125. Further studies on ferredoxin
(Rv0763c) and ferredoxin reductase (Rv3106) expression
S137
REFERENCES
1. Jackson, C.J., Lamb, D.C., Kelly, D.E., Kelly, S.L.:
FEMS Letts 192, 159 (2000).
2. Kelly, S.L., Lamb, D.C., Cannieux, M., Greetham, D.,
Jackson, C., Marczylo, T., Ugochuckwu, C. and Kelly,
D.: Biochem. Soc. Trans. 29, 122 (2001).
3. Jackson, C. J., Lamb, D.C., Marczylo, T.H., Parker,
J.E., Manning, N.L., Kelly, D.E. and Kelly, S.L.: Biochem. Biophys. Res. Comm. 301, (2003).
TP41
CYP 2D6 „INTERMEDIATE
METABOLIZER” PHENOTYPE
AS A CONSEQUENCE OF
GENETICALLY DETERMINED
ALTERNATIVE SPLICING
CLAUDIA CESARO, SEBASTIAN RAIMUNDO,
KATHRIN KLEIN, ELKE SCHAEFFELER,
MICHEL EICHELBAUM, MATTHIAS SCHWAB, and
ULRICH M. ZANGER;
[email protected]
CYP2D6 is responsible for the metabolism of many clinically used drugs including antidepressants, neuroleptics,
beta-blockers and others. CYP2D6 expression is highly polymorphic with more than 70 known alleles which code for
protein products with variant or lacking function1. In Caucasian populations, four phenotypes termed ultrarapid
(UM), extensive (EM), intermediate (IM) and poor metabolizers (PM) are observed. Using liver samples of phenotyped patients, we have shown that these phenotypes are
the result of different protein expression levels2. Our recent
work concentrated on the IM phenotype, which affects
about 10-15% of the population most of whom were shown
to have the genotype *41 / *0, with *41 designating a novel functionally impaired allele and *0 designating any
nullallele3. The *41 allele is genetically distinct from *2 in
a putative NFkappaB binding site at -1584 bp3 but luciferase reporter-gene transfection experiments suggest that
this promoter polymorphism may not explain the large difference in phenotypic expression between *2 and *41. We
therefore undertook a complete sequence analysis of the *2
and *41 alleles comprising the entire upstream region between CYP2D6 and CYP2D7 as well as all introns. Apart
from several novel SNPs which are found in both alleles,
one novel intronic mutation at 2988 (G>A) was found only
in *41 alleles but not in *2. To investigate whether this
intronic mutation may be responsible for the decreased expression of *41, we hypothesized that it may influence
splicing. Using liver RNA samples of genotyped patients,
a specific RT-PCR assay was developed to detect splicing
events between exon 5 and 9, the region where the intron
mutation is located. Individuals with one or two *41 alleles were subsequently shown to lead to an additional PCR
product which was about 150bp shorter than the normal
product. Sequencing of the splice variant revealed the deletion of the entire exon 6. All other genotypes showed
only minute amounts of this splice variant. Because deletion of exon 6 interrupts the open reading frame,the alternative splicing should result in decreased expression of
CYP2D6 protein, thus explaining the IM phenotype of individuals having the *41/ *0 genotype. To further investigate this hypothesis, quantitative analyses of splice variants and protein expression and further correlation studies
between genotype and phenotype are in progress.
REFERENCES
2. Zanger UM, Fischer J, Raimundo R, Stüven T, Evert
BO, Schwab M, Eichelbaum M. Pharmacogenetics 11,
573 (2001)
3. Raimundo S, Fischer J, Eichelbaum M, Griese E-U,
Schwab M, Zanger UM. Pharmacogenetics 10, 577
(2000)
Supported by the German Ministry of Education and
Science and the Robert Bosch Foundation Stuttgart.
TP42
which in the Caucasian population being *2 (28.5%), *4
(19.5%), and *5 (4.1%)1. Previous methods for identifying
the various CYP2D6 genotypes have involved allele-specific polymerase chain reactions (PCR) followed by agarose
gel electrophoresis1-3, which when investigating the presence of multiple variant alleles are time consuming and
expensive. This study aimed at establishing a sequencing
method to determine the presence of multiple variant alleles in a Caucasian population. Following informed consent
of the subjects (n=25) genomic DNA was isolated from either blood samples using a QIAamp DNA mini kit (QIAGEN Pty Ltd, Australia). Initial PCRs using primers previously published2 were performed to produce two templates
isolating regions in exons 3-4: position 1297 to 2034, and
exons 5-6: position 2010 to 3112 (numbers based on translation start). PCR products were purified using a QIAquick
PCR purification kit (QIAGEN) and sequenced using the
following forward primers: position 1556 5’-GTG GGG
CTA ATG CCT TC-3’, and position 2379 5’-GAG ACT
TGT CCA GGT GAA CG-3’. Sequencing reactions were
®
performed with an ABI PRISM BigDye™ Terminator kit,
®
version 3 with analysis on an ABI PRISM 3700 DNA
analyzer (Applied Biosystems, Australia). Using this method we were able to identify CYP2D6 alleles *1 to *4 and
*6 to *9 and the allele frequencies were comparable to
those found previously in a Caucasian population1: *1,
38%; *2, 34%; *3, 0%; *4, 20%; *6, 2%; *7, 0%; *8, 0%;
and *9, 4%. This is the first method to allow genotyping
via sequencing methods, which is a time efficient and reliable alternative to conventional PCR methods enabling information regarding multiple variant alleles to be obtained
rapidly. Further development of the method to allow identification of other variant alleles is ongoing.
REFERENCES
IDENTIFICATION OF CYP2D6
GENOTYPE VIA A NEW
SEQUENCING PROTOCOL
1. Griese E.U., Zanger U.M. et al.: Pharmacogenetics. 8,
15 (1998).
2. Heim M., Meyer U.A.: Lancet. 336, 529 (1990).
3. Johansson I., Lundqvist E. et al.: Proc Natl Acad Sci
U S A. 90, 11825 (1993).
JANET K COLLERa, HEATHER M JAMESb,
DAVID GILLISb, BENEDETTA C SALLUSTIOc, And
ANDREW A SOMOGYIa
a
Department of Clinical and Experimental Pharmacology,
University of Adelaide, SA 5005, Australia, bDivision of
Human Immunology, Institute of Medical and Veterinary
Science, SA 5000, Australia, cDepartment of Cardiology
and Clinical Pharmacology, The Queen Elizabeth Hospital, SA 5011, Australia, [email protected]
The cytochrome P450 (CYP) 2D6 isoform mediates the
oxidative metabolism of many different classes of drugs
used commonly in clinical practice. The clinical consequences of the genetic polymorphism of CYP2D6 have been
extensively reported; between 5-10% of Caucasians classified as poor metabolisers (PM). To date, approximately 75
variant alleles have been identified, the most frequent of
S138
TP43
USE OF 2D - ELECTROPHORESIS AND MALDI - TOF IN THE
IDENTIFICATION OF HEPATIC
CYP1A ISOFORMES OF
PROCHILODUS SCROFA,
A BRAZILIAN FRESHWATER
FISH
DA SILVA M.E.F.a*, SILVA J. A.a, MARTINS D.a,
MARANGONI S.a, NOVELLO J.C.a, and
MEIRELLES N. C.a
a
Laboratory of Biomembranes, Departament of Biochemistry. Institute of Biology - State University of Campinas
P.O. Box - 6109. bLaboratory of Protein Chemistry, Departament of Biochemistry. Institute of Biology - State University of Campinas P.O. Box - 6109. [email protected]
Cytochromes P450 constitute a superfamily of the phase
I enzymes whose primary task is the detoxification of both
endogenous and xenobiotic compounds (Omura, 1999).
Fish, among the non-mammalian species, received great interest because they are direct food source for human as well
as conveyors of toxic chemicals to human beings. The aim
of present study is the identification of hepatic isoformes of
CYP in Prochilodus scrofa, with emphasis in CYP1A, in
a Brazilian fish belonging the family Prochilodontidae and
knowing as Curimbatá that has a high economic value in
southeast region of Brazil. Purification of CYP1A was done
Fig. 1. Tryptic peptide mass fingerprint of CYP1A
S139
by Reverse Phase HPLC on a C18 column. The fractions
collected were applied on a SDS - PAGE 12,5% or in a 2DPAGE. Spots were collected, and treated with Trypsin. CYP
cleavage products were extract with acetonitrille. Peptide
fragmentation were made using MALDI-TOF. Purified
CYP1A was characterized with respect to electrophoretic,
immunochemical and biocatalytic properties. CYP1A fractions produced a band on SDS-PAGE with an apparent molecular weigh of 54kDa. Purified CYP1A of P. scrofa also
showed a strong cross-reactivity with antibody directed
against CYP1A homologue purified from scup. Two major
spots were identified in the 2D-PAGE. They have the same
molecular weight, but pI were 5.5 and 6.0 respectively;
which suggest the presence of 2 isoformes of the same protein, CYP1A1 and CYP1A2. Both spots showed that they
Tab. 1. CYPIA tryptic peptides analyse
Theor. AVG
34571.1+61,26
Sequence
position
1-32
Tryptic
peptide
AV observed
T1
3354,9+1,002
3131.2±82.13
44-73
T2
3103.03±0.648
2849.64+6,49
36-62
T3
2813.50+0.95
2372.5±13.53
409-427
T4
2340.76S±0.93
1804.90+0
194-210
T5
1792,03+0.38
1577,7±14.05
471-483
T6
1585.89±11.45
1375,11+44.80
270-280
T7
1385.64+0.32
1045,86±1869
428-436
T8
1041.42±0.53
851,45
17-177
T9
844.21±1.19
have the same profile of fragmentation, which confirm that
they are isoformes. Tryptic peptide mass fingerprint of
CYP1A purified by HPLC showed the presence of 9 masses
that matched the expected tryptic peptides obtained through
theory digestion of database sequence (Fig. 1). These value
corresponding to 30% of the translated amino acids of
CYP1A family of other fishes (Buhler and Wang-Buhler,1998). Among those peptides, there are two of than that
showed major importance T1, that could represent the NH2terminal of CYP 1A1 and T2, which could represent a more
conservative region (Graham and Peterson, 1999). Table 1
shows CYP1A tryptic peptide analyze by MALDI-TOF. All
those characteristics strongly suggest that this new procedure is efficient to purify simultaneously different isoformes of hepatic CYP1A from P. scrofa, identifying the conservative region of protein.
Supported by FAPESP.
REFERENCES
1. Omura T.: Biochem. Bioph. Res. Comm. 266, 690
(1999).
2. Buhler D. R., Wang-Buhler J. L.: Comp. Biochem. Physiol. 121C, 107 (1998).
3. Graham S. E., Peterson J. A.: Arch Biochem. Biophys.
369, 24 (1999).
TP44
NF-I REGULATES CYP2A5
TRANSCRIPTION IN MOUSE
PRIMARY HEPATOCYTES
vation region was further characterized by Dnase I footprinting. Incubation of the Cyp2a5 proximal promoter region with hepatocyte nuclear extracts protected a single
clear footprint in the studied area centered over a sequence
similar to nuclear factor I (NF-I) binding site. Protein binding to the detected footprint sequence was further analyzed with electrophoretic mobility shift assay. Three retarded protein bound DNA complexes were detected of which
all were abolished by competition with consensus oligo for
NF-I. Mutation of nucleotides critical for NF-I binding eliminated competition. Further, an antibody specific for NFI supershifted one of the complexes. The functional contribution of NF-I site for transcriptional activation of Cyp2a5
gene was confirmed by mutation of the NF-I binding sequence in the Cyp2a5 promoter constructs.
In conclusion, these results indicate that the members
of the NF-I protein family significantly contribute to
Cyp2a5 expression in hepatocytes, and in addition to the
previously suggested role in olfactory mucosa, are also involved in liver-enriched expression of Cyp2a5.
REFERENCES
1. Schrem, H., Klempnauer, J., and Borlak, J. Pharmacol.
Rev. 54, 129 (2002).
2. Su, T., Sheng, J. J., Lipinskas, T. W., and Ding, X. Drug
Metab Dispos. 24, 884 (1996).
TP45
JOHANNA ULVILAa, OLAVI PELKONENa,
MASAHIKO NEGISHIb, and JUKKA HAKKOLAa
a
of Oulu, Oulu, Finland. bPharmaceutical Section, Laboratory of Reproductive and Developmental Toxicity, NIEHS,
National Institute of Health, Reseach Triagle Park, NC
27709, USA. [email protected]
Xenobiotic metabolising cytochrome P450 (CYP) enzymes are expressed in tissue specific manner and the highest levels are detected in the liver. The liver expression is
believed to be controlled mainly by the liver enriched transcription factors1 but few studies have focused on constitutive regulation of individual CYP genes. Cytochrome
P4502a5 (Cyp2a5) gene is expressed principally in liver
and olfactory mucosa2. In the current study, the transcriptional mechanisms of hepatocyte specific expression of
Cyp2a5 were studied in mouse primary hepatocytes.
The Cyp2a5 5’-flanking regulatory region -3033 to +10
was cloned in front of a luciferase reporter gene and transfected into hepatocytes. Deletion analysis of the promoter
revealed two activating sequence regions localized at proximal and distal parts of the promoter. The proximal acti-
S140
CYP2C9 PHENOTYPING USING
LOW-DOSE TOLBUTAMIDE
ALEXANDER JETTERa,
MARTINA KINZIG-SCHIPPERSb, ANDREAS SKOTTb,
ANDREAS LAZARa, DOROTA TOMALIK-SCHARTEa,
MONIKA WALCHNER-BONJEANa,
URSULA HERINGa, HENDRIK LÜCKa,
WAFAÂ JABRANEa, DIRK KASELa, UWE FUHRa, and
FRITZ SÖRGELb.
a
Institute for Pharmacology, Clinical Pharmacology, University of Köln, Gleueler Str. 24, D-50931 Köln, Germany;
b
Institute for Biomedical and Pharmaceutical Research,
Paul-Ehrlich-Str. 19, D-90562 Nürnberg-Heroldsberg,
Germany, e-mail: [email protected]
Tolbutamide (TOL) has been established as a probe for
the assessment of CYP2C9 activity in vivo. However, the
validity and accuracy of the molar ratio of (carboxytolbutamide, CAR + 4-hydroxytolbutamide, 4-OH) / TOL in
urine 6 - 12 h after TOL intake, proposed as a CYP2C9 metric, is unclear.
We therefore analysed plasma and urine samples of 25
healthy male volunteers using LC-MS/MS collected before
and up to 24 h after intake of a single oral dose of 125 mg
TOL in the framework of two low-dose cocktail studies for
phenotyping of important drug-metabolising enzymes.
Subjects fasted before, and up to 6 h after drug intake. The
limits of quantification were 15 and 12.5 ng/mL for TOL,
15 and 25 ng/mL for 4-OH, and 75 and 25 ng/mL for CAR
in plasma and urine, respectively. Genotyping was carried
out by direct sequencing of exon 3 and 7 for identification
of the *2 to *5 alleles, respectively. Pharmacokinetic parameters were calculated with a compartment-free approach,
and one way ANOVA was used for comparison between
the genotypes.
CYP2C9 genotypes were *2/*2 in one, *1/*3 in three,
*1/*2 in six, and *1/*1 in 15 volunteers, and reflected mean
oral TOL plasma clearance (0.57, 0.60 (95% confidence
interval, CI, 0.58 - 0.62), 0.79 (95% CI, 0.66 - 0.91), and
0.85 (95% CI, 0.80 - 0.89) L/h for the four genotypes, respectively). The urinary 6 - 12 h metabolic ratio did not correlate to TOL plasma clearance (r2=0.136, p=0.07) or any of
the pharmacokinetic parameters tested. TOL in plasma samples collected 6 to 24 hours after drug intake correlated significantly to TOL clearance. The best correlation was observed in samples drawn 24 h after TOL intake (r2=0.83,
p<0.0001). The mean 24 h TOL concentrations also reflected
CYP2C9 genotypes: *2/*2, 3.27 µg/mL, *1/*3, 3.13 µg/mL
(95% CI, 2.68 - 3.58 µg/mL), *1/*2, 2.03 µg/mL (95%CI,
1.51 - 2.56 µg/mL), *1/*1, 1.70 µg/mL (95% CI, 1.50 - 1.90
µg/mL). The TOL plasma AUC and the TOL elimination
half-life were well correlated to TOL plasma concentrations
24 h after dosing (r2=0.89 and 0.77, p<0.0001 each). No adverse event caused by TOL was noted.
In conclusion, phenotyping of CYP2C9 can be carried
out with a TOL dose as low as 125 mg, which reduces the
risk of undesired hypoglycaemia caused by TOL. For the
simplification of CYP2C9 phenotyping, it seems more favourable to determine TOL in a single plasma sample taken
24 h after intake of a low 125 mg TOL dose than to determine urinary molar ratios. However, this procedure has to be
validated in a larger population of volunteers and patients.
TP46
CYP2U1, and the corresponding gene was found to be located on chromosome 4. We found an open reading frame
of 1635 nucleotides, which encodes a 544 amino acid long
polypeptide. The predicted amino acid sequence of
CYP2U1 displays up to 39% identity to other human family 2 members, with closest resemblance to CYP2R1.
CYP2U1 is, like CYP2R1, conserved across species, showing a 58% homology to fugu fish and 78% homology to
murine CYP2U1.
One of the structural differences of the CYP2U1 gene
compared to other CYP2 family members is that CYP2U1
consists of only five exons. Furthermore it has a longer
amino acid sequence than other CYP2s. When aligning the
CYP2U1 amino acid sequence to CYP2R1, CYP2D6 and
CYP2C5, it has 40-50 extra amino acids. Approximately
20 of these amino acids are found in the very NH2-terminal, and 25 are located after the NH2-terminal membrane
spanning region.
The mRNA expression of CYP2U1 was examined
using Northern blotting, and revealed high expression levels of CYP2U1 especially in thymus, but also in heart and
brain. The high mRNA expression seen in both adult and
fetal thymus was confirmed using dot blot analysis.
The observed high and specific expression of CYP2U1
in thymus as well as the high conservation across species
might indicate an important endogenous function. The substrate specificity is currently evaluated after cDNA expression in heterologous systems.
TP47
RAPID TRANSLOCATION OF
CYP11A1 INTO YEAST
MITOCHONDRIA HAMPERS
ITS NORMAL SORTING AND
FOLDING
IRINA E. KOVALEVA, LYUDMILA A. NOVIKOVA,
PAVEL A. NAZAROV, SERGEI I. GRIVENNIKOV, and
VALENTIN N. LUZIKOV
A NOVEL THYMUS-SPECIFIC
CYTOCHROME P450, CYP2U1
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University,Russia
[email protected]
MARIA KARLGREN,
and MAGNUS INGELMAN-SUNDBERG
Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm,
Sweden, [email protected]
In the human genome about 55 active cytochrome P450
genes and 26 pseudogenes have been identified. Most of
the corresponding enzymes have been rather well characterized, whereas some, mainly extrahepatic forms, remains
to be characterized. Based on homology searches we identified a novel cytochrome P450 cDNA designated
S141
Topogenesis of cytochrome P-450scc, a resident protein
of the inner membrane of adrenocortical mitochondria, is
still obscure. In particular, little is known about the cause
of its tissue specificity. In an attempt to clarify this point,
we examined the process in Saccharomyces cerevisiae
cells synthesising P-450scc as its native precursor
(pCYP11A1) or versions in which its N-terminal addressing presequence had been replaced with those of yeast mitochondrial proteins: CoxIV1-25 and Su91-112. We found the
pCYP11A1 and CoxIV1-25-mCYP11A1 versions to be effectively imported into yeast mitochondria and subjected to
proteolytic processing. However, only minor portions of
the imported proteins were incorporated into mitochondrial
membranes, whereas their bulk accumulated as aggregates
insoluble in 1% Triton X-100. Along with previously published data1, this suggests that a distinguishing feature of
the import of the CYP11A1 precursors into yeast mitochondria is their easy translocation into the matrix where
the foreign proteins mainly undergo proteolysis or aggregation. The fraction of CYP11A1 that happens to be inserted into the inner mitochondrial membrane is effectively
converted into the catalytically active holoenzyme. Experiments with the Su91-112-mCYP11A1 construct bearing a reexport signal revealed that, after translocation of the fused
protein into the matrix and its processing, the Su967-112 segment ensures association of the mCYP11A1 body with the
inner membrane, but proper folding of the latter does not
take place. Thus it can be said that the most specific stage
of CYP11A1 topogenesis in adrenocortical mitochondria is
its confinement and folding in the inner mitochondrial
membrane. In yeast mitochondria, only an insignificant
portion of the imported CYP11A1 follows this mechanism.
TP49
REFERENCES
1. Savel’ev A.S., Novikova L.A., Kovaleva I.E., Luzikov
V.N., Neupert W., Langer T.: J. Biol. Chem, 273, 20596
(1998).
TP48
tracellular proteolysis of the three-component fusions depend on the origin and arrangement of their constituents.
To shed some light on the assembly of catalytic domains in
the fused molecules, we analysed catalytic properties of
various two-component fusions (P450scc-Adx, AdxP450scc, and AdR-Adx). Their ability to carry out sidechain cleavage reaction in the presence of corresponding
missing component of the whole cholesterol hydroxylase/lyase system and the dependence of this reaction on
the presence of added individual components of the double
fusions themselves were in focus. This analysis has revealed that active centres in the double fusions are either separated from each another or somehow deformed; in some
cases they were unreachable for exogenous partners. In total, our data suggest that upon folding of polypeptide chains including P450scc, Adx, and AdR, corresponding catalytic domains are not formed independently of each other,
i.e., in a way that each domain assumes its normal structure
with any arrangement of others.
P450 ALLELE AND HAPLOTYPE
FREQUENCY STUDY IN
DIFFERENT POPULATIONS
USING THE CODELINK™
SNP ASSAY
R. BONNER, B. CHUI, M. GASKIN, P. GWYNNE,
A. LEDESMA, T. PECK, T. PETERS, A. SILBERGLEIT,
M. AMJADI, R. FELDMAN, and C. YAMASHIRO
SYNTHESIS AND PROPERTIES
OF FUSED PROTEINS
COMPOSED OF COMPONENTS
OF THE CHOLESTEROL
SIDE-CHAIN CLEAVAGE
ENZYME SYSTEM
Amersham Biosciences, 3200 West Germann Road, Chandler, AZ 85248, [email protected]
PAVEL A. NAZAROVa, VALERII L. DRUTSAa,
WALTER L. MILLERb, VLADIMIR M. SHKUMATOVc,
VALENTIN N. LUZIKOVa,
and LYUDMILA A. NOVIKOVAa
a
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Russia; bDepartment of
Pediatrics and The Metabolic Research Unit, University of
California, San Francisco, USA; cInstitute of PhysicoChemical Problems, State University, Minsk, Belarus
[email protected]
We studied the properties of various fused combinations of the components of mitochondrial cholesterol hydroxylase/lyase, including cytochrome P450scc, adrenodoxin
(Adx), and adrenodoxin reductase (AdR). To this end, recombinant DNAs encoding these constructs were expressed in Escherichia coli cells. We have found that both cholesterol side-chain cleavage activity and sensitivity to in-
S142
It is now evident that a significant part of variation in
drug efficacy and safety is of a hereditary nature, and can
be traced down to polymorphisms in genes involved in
drug metabolism. Establishing the correlations between
drug metabolizing phenotypes and P450 genotypes and
haplotypes will make clinical trials safer and provide the
next step towards a personalized medicine.
CodeLink™ SNP bioarrays from Amersham Biosciences are robust tools for broad-based single nucleotide polymorphism (SNP) genotyping, which can be used in linkage
mapping or candidate gene profiling. The assay process is
simple, straightforward, and highly efficient with automation allowing for high throughput genotyping. Data analysis using the CodeLink software provides rapid data processing yielding genotyping reports and other analysis capabilities. The combination of internal controls and software features allows for efficient data validation.
For SNP detection, CodeLink platform uses enzymatic
allele specific extension (ASE) of oligonucleotide probes.
In CodeLink SNP P450 assay, a template for ASE is generated by highly specific long-range multiplex PCR follo-
wed by amplicon purification and fragmentation. Unique
proprietary PCR primer design ensures absolute specificity
of amplification despite of high DNA homology within
P450 gene subfamilies.
CodeLink SNP P450 v.2.0 allows for genotyping of 130
SNPs in nine genes: CYP1A1, CYP1A2, CYP1B1, CYP2C19,
CYP2C9, CYP2D6, CYP2E, CYP3A4, and CYP3A5.
We sequenced 46 samples from three major populations
at these loci, and sequence data was used as a „gold standard“ to estimate assay accuracy.
From Coriell Human Diversity panels, 230 samples representing three major populations were genotyped and population-specific allele and inferred haplotype frequencies
have been calculated.
TP50
ASSOCIATION OF GENETIC
POLYMORPHISMS IN CYTOCHROME P450 2E1 AND OTHER
BIOTRANSFORMATION
ENZYMES WITH PROGRESSION
OF LYMPHOMAS
ents with NHL (P = 0.024), and especially that of aggressive
diffuse NHL (P = 0.007). Grading of NHL seemed to be associated with this polymorphism as well (P = 0.041). The
EPHX1-exon 3 genotype distribution was significantly different between control males and males with all lymphomas
(P = 0.01) or with NHL (P = 0.019). The variant homozygous
genotype of GSTP1-exon 5 was prevalent in all MH
(OR = 2.08, 95% CI = 1.05 - 4.14, P = 0.035) and this difference was particularly evident in females (OR = 2.97, 95%
CI = 1.16 - 7.61, P = 0.023). A significant difference in the
distribution of GSTP1-exon 5 genotypes was found between
NHL tumors larger vs. smaller than 5 cm (P = 0.03). In conclusion, the results suggest that genetic polymorphisms of
biotransformation enzymes may play a significant role in the
development and progression of lymphoid malignancies.
The work at this project was supported by grant IGA
6747-3.
TP51
PAVEL SOUâEK, JANA ·ARMANOVÁ,
SIMONA ·ÒSOVÁ, and IVAN GUT
Biotransformations Group, Center of Occupational Diseases, National Institute of Public Health, ·robárova 48,
Praha 10, 10042 Czech Republic, e-mail: [email protected]
The genetically variable biotransformation enzymes:
cytochromes P450 (CYP, EC 1.14.14.1), epoxide hydrolase
(EPHX1, EC 3.3.2.3), NAD(P)H:quinone oxidoreductase
(NQO1, EC 1.6.99.2), and glutathione S-transferases
(GST, EC 2.5.1.18) metabolize and conjugate drugs, carcinogens, and natural products. In addition, high number of
human cancer cases result from exposure to environmental
carcinogens suggesting that individual effectiveness in the
detoxification of these chemicals may influence susceptibility to malignant disease. The aim of our study was to determine frequencies of the above listed polymorphisms of
biotransformation enzymes in healthy population of the
Czech Republic and to compare these frequencies with
data on group of lymphoma patients. Polymerase chain reaction-restriction fragment length polymorphism based genotyping assays were used to determine the frequency of
polymorphisms in CYP1A1 (3’-flanking region), CYP2E1
(5’-flanking region and intron 6), EPHX1 (exon 3 and exon
4), NQO1 (exon 6), GSTM1 (deletion), GSTP1 (exon 5),
and GSTT1 (deletion) in a case-control study comprised of
219 patients with morbus Hodgkin (MH) and non-Hodgkin’s lymphomas (NHL) and 455 age- and sex-matched
healthy individuals. The distribution of genotypes of
CYP2E1-intron 6 was significantly different between the
control group and that of all lymphomas (P = 0.03), pati-
S143
GENOME-WIDE EXPRESSION
PROFILING AND XENOBIOTIC
INDUCIBILITY OF P450
MONOOXYGENASE GENES
IN THE WHITE ROT FUNGUS
PHANEROCHAETE
CHRYSOSPORUM
JAGJIT S. YADAV, and H. DODDAPANENI
Molecular Toxicology Division, Department of Environmental Health, University of Cincinnati College of Medicine,
Cincinnati, OH 45267-0056, USA, [email protected]
Phanerochaete chrysosporium belongs to white rot
fungi which have the unique ability to biodegrade lignin
(plant aromatic polymer), a rate-limiting step in the nature’s carbon cycle, and a broad specificity to oxidize and
mineralize (to CO2) a wide range of environmental chemical pollutants. P. chrysosporium has been widely used as
a model to study the physiology, biochemistry, and genetics of biodegradation of lignin and chemical pollutants by
white rot fungi. Lignin degradation occurs under nutrientlimited conditions when the fungus enters secondary metabolism, whereas chemical pollutants are degraded under
both nutrient-limited (ligninolytic) and nutrient-rich (nonligninolytic) culture conditions. Recognized role of P450
monooxygenases in these environmentally and biotechnologically significant biodegradation processes has led to an
increasing interest in the characterization of cytochrome
P450s in this organism.
In an effort to characterize P450 monooxygenase systems of P. chrysosporium, we cloned the first two fulllength P450 monooxygenase genes pc-1 and pc-2 belonging to the CYP63 family1, 2 and the P450 reductase gene3.
Subsequently, whole genome sequencing of this organism
by the Joint Genome Institute of the US Department of
Energy led to the identification of the presence of over 150
P450 genes, of which 100 genes have been assembled fulllength (http://drnelson.utmem.edu/nelsonhomepage.html).
Except for the conserved fungal P450 genes CYP51 and
CYP61 and the three CYP63 genes (CYP63 A1, A2, and
A3) experimentally cloned in our laboratory, other P450
genes in P. chrysosporium genome have unknown identity
as they do not bear significant homology to the known
P450 genes indicating a unique CYPome of this organism.
Based on our phylogenetic analysis, the 150 genes are
groupable into 26 clusters, most of them with multiple
members. The cloned CYP63 members (CYP63A1, A2,
A3) belong to one of these clusters.
Functional characterization of the P450 genes revealed
in the white rot fungal genome would require identification
of their substrates and the physiological conditions regulating their expression. In this regard, we developed a custom
P450 microarray and studied genome-wide expression of
87 P450 genes representing all 26 clusters under varying
physiological states. A total of 66 genes showed expression
under both nutrient-limited and nutrient-rich culture conditions, of which 19 and 13 genes were upregulated (2- to 12fold) under the two physiological states, respectively. We
investigated xenobiotic inducibility of the P450s in response to 35 individual chemicals belonging to aromatic/polyaromatic, alicyclic, and aliphatic groups. CYP63A1
(pc-1) and CYP63A3 (pc-3) showed several fold (2 to 11fold) induction by alkyl-substituted aromatics and oxygenated aromatic and aliphatic xenobiotics. The results yielded information on potential substrates and nutritional regulation for the individual P450 genes tested. Additional
information from our on-going studies on genome-wide
expression and induction on other P450s in the white rot
fungus will also be presented.
REFERENCES
1. Yadav J.S., Loper J.C., Soellner M.B.: 101st ASM Meeting, Orlando, FL, 20-24 May, 2001. Abstract Book,
Q-469.
2. Yadav J.S., Soellner M.B., Loper J.C., Mishra P.K.:Fungal Genet. Biol.38, 10 (2003).
3. Yadav J.S., Loper J.C.: Curr. Genet. 37, 65 (2000).
S144
TP52
HAPLOTYPE ANALYSIS
OF CYTOCHROME P450 2B6
– LINKAGE BETWEEN
PROMOTER- AND EXONIC
MUTATIONS.
JÖRG ZUKUNFTa, THOMAS LANGb, KATHRIN
KLEINa, TANJA RICHTERa, MATTHIAS SCHWABa,
MICHEL EICHELBAUMa, and ULRICH M. ZANGERa
a
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstr. 112, 70376 Stuttgart, Germany,
[email protected], bEpidauros Biotechnology
AG, Bernried, Germany
The human cytochrome P450 2B6 (CYP2B6) isoenzyme
is a highly polymorphic and inducible member of the CYP
family. We have previously shown that some exonic mutations are associated with changes in expression in human liver
but the underlying mechanism remained unclear. We therefore speculated that identification of haplotypes considering
promoter and exonic mutations would be more informative
than individual mutations. Using our liverbank, a total of
98 individuals were genotyped for 15 dimorphic sites. Ten of
these SNPs (-1848C>A, -1778A>G, -1578C>A, -1489G>A,
-1456T>C, -1186G>C, -801G>T, -750T>C, -591A>G and
-82T>C) are positioned in the promoter region whereas the
remaining 5 (64C>T, 516G>T, 777C>A, 785A>G and
1459C>T) lead to amino acid changes in the coding sequence. Three different kinds of phenotypic data are available
for the examined liver samples: CYP2B6 mRNA content
quantitated by TaqMan RT-PCR, protein content determined
by Western Blotting, and CYP2B6 activity, measured by hydroxylation of the specific probe drug bupropion in liver microsomes. A haplotype analysis was conducted using the software package Arlequin3. Twentyone different haplotypes
were calculated, and strong linkage disequilibrium between
promoter SNPs (-1456T>C, -750T>C) and non-conservative
exonic mutations (516G>T, 785A>G) was observed.
Our population sample was fragmented into more than
20 inferred genotypes which comprized between 1 and 12
individuals. Linkage between promoter- and exonic mutations could give a possible explanation for significantly decreased CYP2B6 protein content associated with some of
the previously described alleles2. However, by analyzing
expression data between different genotypes using
ANOVA no statistically significant relationships were
found, although there were marked differences between
groups. This result is probably due to the small number of
individuals in each group. It appears, therefore, that in the
case of highly polymorphic genes like CYP2B6 complete
haplotype analysis may lead into a statistical trap. For reasons of practicability it may be necessary to reduce the
number of subgroups by merging functionally equivalent
genotypes for genotype-phenotype correlation analysis.
(grant ZA 245/2-1) and the Robert Bosch Foundation,
Stuttgart.
condary structure prediction of mRNA at RBS is a useful
approach to control the expression level of heterologous
protein in E. coli cells.
REFERENCES
REFERENCES
Hofmann U, Eichelbaum M, Schwab M, Zanger UM.
Pharmacogenetics 11, 399 (2001).
3. Arlequin - a software for population genetics data analysis. http://lgb.unige.ch/arlequin.
1. Gegner, J.A., Dahlquist, F.W.: Proc. Natl. Acad. Sci.
USA 88, 750 (1991)
2. Kimura, S., Emi, Y., Ikushiro, S., Iyanagi, T.: Biochim.
Biophys. Acta 1430, 290 (1999)
3. Kimura, S., Kawamura, M., Iyanagi, T.: J. Biol. Chem.
278, 3580 (2003)
TP53
TP54
HIGH-LEVEL EXPRESSION OF
PORCINE P450 REDUCTASE
SOLUBILIZED DOMAIN IN
ESCHERICHIA COLI BY
MODULATING THE LOCAL
SECONDARY STRUCTURE OF
MRNA
SHIGENOBU KIMURA, and TAKASHI IYANAGI
INTERACTION OF
CONSTITUTIVE ANDROSTANE
RECEPTOR-RETINOIC X
RECEPTOR (CAR-RXR)
HETERODIMERS WITH
ELEMENTS OF THE CYP2B2
PHENOBARBITAL RESPONSE
UNIT
MARIE-JOSÉE BEAUDET, AMAURY LACHAUD, and
ALAN ANDERSON
Department of Life Science, Graduate School of Science,
Himeji Institute of Technology, Kouto 3-2-1, Kamigori,
Hyogo 678-1297 Japan, [email protected]
The high-level expression of heterologous protein in
E. coli cells is necessary for in vitro study of the protein
using site-directed mutagenesis. However, the expression
of the protein depends on the individual protein, and various problems have to be often overcome to realize its highlevel expression. In this study, direct expression plasmids
for the solubilized domains of NADPH-cytochrome P450
reductase (sCPR) from porcine (PsCPR) and rat (RsCPR)
were constructed using pCWori+ 1 in a similar manner previously described.2,3 PsCPR was minimally expressed,
whereas RsCPR was highly expressed. The substitution of
the nucleotides encoding Thr60Ser61Ser62 in PsCPR with
those of Ala60Pro61Pro62 in RsCPR markedly increased the
expression level. The local secondary structures of mRNA
predicted with a program GeneBee (http://www.genebee.msu.su) suggested that the intramolecular base-pairing
at the ribosome binding site (RBS) in mRNA affected the
protein expression. Silent mutations were systematically
introduced into the codons of Thr60Ser61 in PsCPR to modulate the base-pairing. The expression levels of the silently mutated PsCPRs depended on the predicted local secondary structures of mRNA at RBS. The wild-type
PsCPR, in which Thr60Ser61 was encoded by ACTTCT, was
purified. The purification yield was 45.8 mg/liter of culture
fluid. These results suggest that the introduction of silent
mutation into the N-terminal region based on the local se-
S145
[email protected]
Hepatic cytochrome P450s (CYPs) play a critical role
in the metabolism of hydrophobic xenobiotics and many
are also selectively inducible by xenobiotics. Much progress has recently been made toward understanding the
molecular mechanisms underlying phenobarbital (PB) inducibility of the homologous rat CYP2B1 and CYP2B2 genes, the mouse Cyp2b10 gene and the chicken CYP2H1
gene. A multicomponent 163-base pair enhancer in the 5’
flank of the rat CYP2B2 gene confers PB inducibility on
homologous and heterologous promoters and constitutes
a PB response unit (PBRU)1,2. The PBRU contains three
putative nuclear receptor (NR) binding sites, NR13, NR23
and NR34. Within the PBRU there is a 51-base pair PB response enhancer module (PBREM) which contains the the
NR1 an NR2 sites3. When the PBREM is placed directly
adjacent to the heterologous tk promoter it confers maximal PB responsiveness3, but in the natural sequence context the full PBRU is required to confer a maximal response5. The constitutive androstane receptor (CAR), in the
form of a heterodimer with the retinoid X receptor (RXR),
binds to the retinoic acid ß2 response element (ß-RARE) 6
and to the NR1, NR2 and NR3 sites of the PBRU4,7 and
activates NR1-driven transcription of reporter genes in cell
lines4. CAR-RXR binding to the NR sites is thought to play
a key role in conferring PB responsiveness8. Individual
base pairs of the NR sites were changed and the effects on
CAR-RXR binding, as determined by gel shift analyses,
were assessed. The CAR and RXR proteins were synthesized from expression vectors using coupled in vitro transcription and translation. The same mutant sequences were
incorporated into the NR sites within the PBRU and their
effects on conferring PB responsiveness in luciferase reporter gene assays after transfection into primary rat hepatocytes were evaluated. The results suggest that something
other than, or in addition to, CAR-RXR binding to the NR
sites is required for maximal PB responsiveness.
REFERENCES
263 (1995).
2. Stoltz C., Vachon M.-H., Trottier E., Dubois S., Paquet
3. Honkakoski P., Moore R., Washburn K.A., Negishi M.:
Mol. Pharmacol. 53, 597 (1998).
4. Kim J., Min G., Kemper B.: J. Biol. Chem. 276, 7559
(2001).
Biol. Chem. 275, 38427 (2000).
6. Baes M., Gulick T., Choi H.S., Martinoli M.G., Simha
D., Moore D.D.: Mol. Cell Biol. 14, 1544 (1994).
7. Tzameli I., Pissios P., Schuetz E.G., Moore D.D.: Mol.
Cell Biol. 20, 2951 (2000).
TP55
THE ANTICANCER DRUG
ELLIPTICINE ACTS AS AN
INDUCER OF CYTOCHROMES
P450 1A1/2 AND POTENTIATES
ITS OWN PHARMACOLOGICAL
EFFICIENCY
more efficient metabolite(s) forming DNA adducts1,2. This
implies the importance of several CYPs in producing these
more active antitumor metabolite(s). The aim of the present work is to study whether ellipticine could influence
the expression of the major CYPs oxidizing this drug.
In order to investigate the induction of cytochromes
P450 by ellipticine, Wistar male and female rats, suitable
animal models mimicking the ellipticine activation in humans3, were treated with 4, 40 and 80 mg ellipticine per kg.
Because ellipticine was found to bind to the Ah receptor,
which is known to be responsible for the induction of several enzymes including CYP1A1/2, we examined the expression levels of these CYPs in rat livers. In addition, the
effect of ellipticine on expression of the CYP2B1/2, 3A1/2
and 2E1 enzymes was investigated. Selective antibodies
against rat CYP1A1 and 3A1 and rabbit CYP2B4 and 2E1
were utilized for this study. The expression of CYP1A1/2
proteins in rat livers of both sexes was strongly induced ellipticine. The expression levels of these enzymes in treated
rats are more than one order of magnitude higher than
those in control animals. In analog, the increase of
CYP1A1/2 expression correlates with an increased EROD
activity, a marker for CYP1A1/2, and with that of MROD,
a marker for CYP1A2, and with the oxidation of Sudan I,
a marker for the CYP1A1 activity. The CYP1A1/2 induction is strongly dependent on the dose of ellipticine administered to the rats. The induction is transient, i.e. in the absence of ellipticine, the amount and activity of the induced
CYP1A1/2 decreased to the basal level two weeks after exposure.
The results indicate that a long-term treatment of humans with ellipticine might stimulate its pharmacological
efficiency against cancer diseases, if the CYP induction
also occurs in the target organs of therapy, e.g. the breast.
REFERENCES
DAGMAR AIMOVÁa, TEREZA DLOUHÁa,
EVA FREIb, and MARIE STIBOROVÁa
a
University, Albertov 2030, 128 40 Prague 2, The Czech Republic, [email protected], [email protected], bDepartment of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280, 61920 Heidelberg,
Germany
Ellipticine is an alkaloid exhibiting significant antineoplastic and anti-HIV activities. Cytochromes P450 (CYPs)
are believed to be the major enzymes catalyzing the metabolism of ellipticine. We described that CYP-dependent
metabolism of ellipticine leads to activation of this agent to
S146
1. Stiborová M., Bieler C.A., Wiessler M., Frei E.: Biochem. Pharmacol. 62, 1675 (2001).
2. Frei E., Bieler C.A., Arlt V., Wiessler M., Stiborová M.:
Biochem. Pharmacol. 64, 289 (2002).
3. Stiborová M., Stiborová-Rupertová M., Bofiek-Dohalská L., Wiessler M., Frei E.: Chem. Res. Toxicol. 16, 38
(2003).
TP56
NEWLY-IDENTIFIED
TRANSCRIPTION FACTOR
BINDING SITES IN THE RAT
CYP2B2 PHENOBARBITAL
RESPONSE UNIT : HNF4 AND
PBX-PREP1
TP57
RABBIT CYP4B1 ENGINEERED
FOR HIGH-LEVEL EXPRESSION
IN E. COLI: LIGAND STABILIZATION AND PROCESSING OF
THE N-TERMINUS AND HEME
PROSTHETIC GROUP
MATTHEW J. CHEESMANa, BRIAN R. BAERa,
ELIZABETH M. J. GILLAMb and ALLAN E. RETTIEa
MARIE-JOSÉE BEAUDET, and ALAN ANDERSON
[email protected]
The CYP2B1 and CYP2B2 genes are dramatically induced in rat liver by phenobarbital (PB). A multicomponent enhancer confering PB responsiveness, the PB response unit
(PBRU), is located between -2317 bp and -2155 bp in the
CYP2B2 5’ flanking region1,2. The PBRU contains three nuclear response elements (NR1, NR2 and NR3) that contribute
to conferring PB responsiveness, plus other elements including an HNF4 binding site detected in this study. Mutational
analysis of the HNF4 element was undertaken, using a reporter construct with the PBRU in the natural sequence context
transfected into primary rat hepatocytes. The results suggest
that HNF4 acts as a negative regulator by competing for binding to the NR1A half site thus leading to obstruction of the
nuclear receptor or other activator that is presumed to activate transcripton via the NR1 element. Electrophoretic mobilty shift analysis (EMSA) and super shift analysis of the
effects of mutant sequences on HNF4 binding revealed that
HNF4 binds to the 5’-AGTACAGAGTCTGTG-3’ sequence
of the PBRU. At the NR2 site, mutation of the 4-bp spacer
led to the loss of a prominent doublet in EMSA assays. Inspection of the NR2 spacer and NR2B half site revealed the
presence of a putative binding site for Pbx-Prep heterodimers3. EMSA analysis using the wild type NR2 labeled oligo
and antibody supershift experiments were thus undertaken to
identify the proteins responsible for the characteristic doublet. The proteins were found to be Pbx1b-Prep1 and Pbx2Prep1 heterodimers. This conclusion was confirmed by coupled in vitro transcription/translation analyses. The Pbx-Prep
binding site was defined as 5’-TGACTGACAC-3’; it overlaps the NR2 spacer and the NR2B half site. The possible roles of HNF4 and PBx-Prep1 as negative regulators of PB responsiveness are presently under investigation.
REFERENCES
263 (1995).
3. Berthelsen J., Zappavigna V., Mavilio F., Blasi F.:
EMBO J. 17, 1423 (1998).
S147
a
Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195, and bDepartment of
Physiology and Pharmacology, School of Biomedical Sciences, University of Queensland, St. Lucia 4072, Australia
[email protected];
CYP4B1 is a largely extrahepatic form of cytochrome
P450 that bioactivates numerous xenobiotics including
4-ipomeanol, 3-methylindole and several polyaromatic
amines. CYP4B1 is also interesting from a structural perspective because this isoform, along with several others in
the CYP4 family, possesses a covalently-bound heme prosthetic group. The unique environment of the CYP4B1
active site coupled to its proclivity for xenobiotic bioactivation make CYP4B1 an especially interesting target for
detailed structural studies that will inevitably require significant re-engineering of the enzyme. Therefore, the fulllength, wild-type, rabbit CYP4B1 and three N-terminally
modified genes were cloned into the pCWori+ vector and
expression studies were initiated in E. coli. No spectrally
detectable enzyme was observed from expression of the native sequence. However, incorporation of MALLLAVF as
the first 8 residues (pCW4B1#1) or truncation of 17 residues from the N-terminus (pCW4B1#7) provided > 450
nmoles of membrane-bound, holo-CYP4B1 per liter culture. A third construct, in which the MALLLAVF sequence
was aligned at amino acid residues 14-21 (pCW4B1#3), severely inhibited bacterial cell growth, resulting in very low
yields of the expressed holo-enzyme. CYP4B1#1 and
CYP4B1#7 were isolated in high purity following nickelaffinity and hydroxyapatite chromatography. Solubilization
from bacterial membranes led to substantial converision to
P420 which could be prevented by the addition of α-naphthoflavone as a stabilizing ligand. Mass spectrometry analysis
and Edman sequencing revealed evidence of differential
N-terminal post-translational processing of CYP4B1#1 and
CYP4B1#7. Furthermore, both enzymes were found to
covalently bind greater than 99.5% of their heme prosthetic
group. The fully covalently-linked hemoproteins exhibited
similar rates and regioselectivities of lauric acid hydroxylation to that observed previously for the partially heme processed enzyme expressed in insect cells. These studies
establish conditions for the facile, high level expression of
CYP4B1 in E. coli and demonstrate that the fully, hemecovalently linked enzyme is catalytically active.
TP58
REFERENCES
TRANSCRIPTIONAL REGULATION OF THE CHOLESTEROL
BIOSYNTHETIC PATHWAY IN
THE MALE GONAD
1. H. Wang, F. Liu, C.F. Millette, D.L. Kilpatrick, Mol.
Cell Biol. 22, 24, (2002).
K. FON TACER, and D. ROZMAN
TP59
1Institute of Biochemistry, Medical Centre for Molecular
Biology, Faculty of Medicine, University of Ljubljana,
SI-1000 Ljubljana, Slovenia, [email protected]
Lanosterol 14α-demethylase (CYP51) is the only cytochrome P450 involved in the housekeeping pathway of
cholesterol biosynthesis. It is regulated at the level of transcription by transcription factors of the sterol regulatory
element-binding protein (SREBP) family, in a similar manner as other cholesterogenic genes. Expression of cholesterogenic genes in somatic cells generally depends on cholesterol levels. In contrast to somatic cells, where the goal
is to produce cholesterol, it was proposed that SREBPs do
not play a significant role in transcriptional regulation in
male germ cells, where cholesterol biosynthesis intermediates with signalling properties (sterols MAS) accumulate
and the cAMP-dependent signalling cascade predominates.
Male germ cells do not grow in a primary culture, which is
a big limitation in experimental approaches. We have thus
developed an ex vivo system, with the aim to simulate the
cholesterol biosynthesis conditions in the testis. The level
of transcription factors SREBP in human choriocarcinoma
cell line JEG-3 was reduced by growing cells in cholesterol/fatty acid rich media. The trans-activation by the
cAMP-dependent pathway was achieved by overexpressing transcription factor CREB. The simulation was only
partially successful. cAMP-stimulation in cholesterol-repressed conditions lead to a decrease in cholesterol quantity, which is in accordance with metabolism of male germ
cells that are not efficient in producing cholesterol de novo.
In contrary to expectations, activation of the cAMP-dependent pathway did not result in a change of sterol composition and quantity. Recently, a soluble, 55 kDa cholesterolinsensitive form of SREBP2 (SREBP2gc) was discovered
in male germ cells, which opened a new venue in understanding biochemical processes in the male gonad. To explore this issue, we have measured the level of SREBP proteins has in male germ cells of fasted and normally fed
mice. Interestingly, three SREBP2-immunoreactive proteins (72, 63 and 55 kDa), that are not present in mouse liver
nuclei, have been observed. The 55 kDa protein is likely
the previously identified SREBP2gc, while the other two
SREBP2 isoforms are novel and seem also to be insensitive
to the level of cholesterol. The role of cholesterol-independent forms of SREBP2 on transcriptional regulation of
CYP51 is currently investigated. It seems possible that
cholesterol-independent forms of SREBPs together with
cAMP-dependent stimuli, contribute to accumulation of
cholesterogenic intermediates, the signalling sterols MAS.
S148
CONSTITUTIVE ANDROSTANE
RECEPTOR-RETINOIC X
RECEPTOR (CAR-RXR)-DEPENDENT TRANSCRIPTIONAL
ACTIVATION BY THE CYP2B2
PHENOBARBITAL RESPONSE
UNIT VARIES DRAMATICALLY
WITH DIFFERENT REPORTER
CONSTRUCTS
MAUD GRAVEL, MARC DESROCHERS,
ÉRIC TROTTIER, and ALAN ANDERSON
[email protected]
Hepatic cytochrome P450s play a key role in xenobiotic
metabolism. Some forms are expressed constitutively and
others are inducible. In rat liver, phenobarbital (PB), well
known for inducing CYP2B forms, produces a strong and rapid transcriptional activation of the CYP2B1 et CYP2B2 genes. A PB response unit (PBRU) is localized about 2.2 kb
upstream of the CYP2B2 transcriptional start site1,2. The
PBRU confers PB responsiveness on the CYP2B2 gene as
well as on reporter constructs and possesses the characteristics of a multicomponent transcriptional enhancer2. The
PBRU contains three putative nuclear receptor (NR) binding
sites, NR13, NR23 and NR34. The constitutive androstane receptor (CAR), is required for PB responsiveness in vivo5
and, in the form of a heterodimer with the retinoid X receptor (RXR), binds to the the NR1, NR2 and NR3 sites of the
PBRU and activates NR1-driven transcription of reporter
genes in cell lines. The glucocorticoid receptor interacting
protein 1 (GRIP1) mediates PB-dependent nuclear translocation and activation of CAR in vivo but has been reported
to have only modest effects in vitro6. Furthermore, normal
PB responsiveness of CYP2B genes is not maintained in any
known cell line. In an effort to understand the molecular mechanisms whereby PB induces CYP2B genes in vivo but not
in cultured cells of hepatic origin, expression vectors for
CAR, RXR and GRIP1 were cotransfected into HepG2 cells
along with luciferase reporter constructs carrying the PBRU
in different sequence contexts. CAR activated reporter gene
transcription when the PBRU was juxtaposed to a basal promoter, but was essentially inactive when the PBRU was tested in the natural sequence context. GRIP1 dramatically inc-
reased CAR activation when tested with reporter constructs
in which the PBRU was adjacent to the basal promoter and
also, but to a much lesser degree, when the PBRU was in its
natural sequence context. It may be that HepG2 cells lack as
yet unidentified transcription factors that are necessary, in
the natural sequence context, for the PBRU to confer PB responsiveness on the luciferase reporter.
This research was supported in part by INTAS grant
2001-0596.
TP61
REFERENCES
263 (1995).
Mol. Pharmacol. 53, 597 (1998).
(2001).
5. Wei P., Zhang J., Egan-Hafley M., Liang S., Moore
D.D.: Nature 407, 920 (2000).
6. Min G., Kemper J.K., Kemper B.: J Biol. Chem. 277,
26356 (2002).
TP60
tional-active recombinant P450s in plants using plant viral
vectors.
EXPRESSION OF MAMMALIAN
MICROSOMAL P450S IN PLANTS
USING PLANT VIRUS VECTORS
JGOUN, A. A.a, ELDAROV, M. A.a,
PETUSHKOVA, N. A.b, NOVIKOV, V. K.c,
DOROKHOV, Y. L.c, ATABEKOV, I. Gc,
and SKRYABIN, K. Ga
a
Center ‘Bieonegineering’ Russian Academy of Sciences,
117312, Prospekt 60-let Oktyabrya, 7/1, Moscow, Russia,
[email protected], bInstitute of Biomedical Chemistry, RAMS,
117984, Pogodinskaya 10, Moscow, Russia, [email protected],
c
Department of Virology, Moscow State University, 119899,
Vorob’evy Gory, Moscow, Russia
The P450 enzymes play important roles in plant physiology. The functional genomic and proteomic analysis of
both known and newly discovered plant P450s should rely
on a system for rapid and high-level production of these
proteins in plants. To evaluate the potential of plant viral
vectors for these purpose human CYP1A2 and rabbit
CYP2B4 (both HIS-tagged) were expressed using potatovirus X (pVX201) and tobacco-mosaic (TMV70) vectors.
The transfected N.bentamiana and N.clevelandii plants
showed accumulation of CYP1A2 and CYP2B4 in microsomal fractions and displayed increased 7-MROD and
7-PROD activities respectively. The TMV-based vector
had additional advantages of increased expression level,
insert stability and decreased chlorosis as compared to
pVX201 vector. This data justifies the production of func-
S149
EXPRESSION OF LEUKOTRIENE
B4 ω-HYDROXYLASE IN
HUMAN LEUKOCYTE
HIDEYUKI KUNISHI, YUKO MASUMOTO,
MICHIE EYA, JUNICHI OHTA, SHIHO NISHIMOTO,
KIYOFUMI NAKATA, and YASUSHI KIKUTA
Department of Applied Biological Science, Fukuyama University, Gakuencho-1 Fukuyama, Hiroshima, Japan,
[email protected]
Leukotriene B4 (LTB4), a product of the 5-lipoxygenase-dependent arachidonic acid metabolism, plays a role
as the most powerful mediator of inflammation. In human
neutrophils, LTB4 is rapidly converted into biologically
less active products by the ω-oxidation pathway. The LTB4
ω-hydroxylase, which catalyzes the first step of this pathway, is classified as the CYP4F subfamily. Five
CYP4F isoenzymes have been found in human tissues.
CYP4F3, an enzyme in human neutrophils, catalyzes
ω-hydroxylations of various eicosanoids and related compounds as well as LTB4. However, the Km value for LTB4
(0.64 µM) is much lower than those for other substrates.
Although LTB4 ω-hydroxylase was found in human neutrophils first, the expression of CYP4F isoenzymes in other
leukocytes was not investigated well. In this study, we have
analyzed the expression of CYP4F enzyme in human peripheral leukocytes and human myeloid leukemia cell line
HL60 by immunocytochemistry.
Homogenates of the neutrophil lich fraction of human
leukocytes catalyzed LTB4 ω-hydroxylation activity
(23.4 pmol/min/mg protein), and those of the monocyte lich
fraction showed 11 times lower activity (2.1 pmol/min/mg
protein). No activity was detected in the lymphocyte lich
fraction. However, neutrophils contaminated into monocyte fraction can catalyze this activity. Then the expression
of the CYP4F enzymes in human leukocytes was examined
by immunocytochemistry. Western blot analysis with the
antibody against CYP4F3 peptide showed the presence of
a protein with the same apparent molecular weight as
CYP4F3 in human neutrophil microsomes. When this antibody was used for the immunocytochemistry, fluorescence
was observed in the cytoplasm of human leukocytes. Most
of these positive cells had segmented nuclei. The double
staining with anti-CD14 and anti-CYP4F3 antibodies showed that one third of CD14 positive cells, monocytes, were
also stained by anti-CYP4F3 antibody. HL60 cells showed
no LTB4 ω-hydroxylase activity, and the cells stimulated by
all-trans-retinoic acid (RA) for 6 days catalyzed the LTB4
ω-hydroxylation. HL60 cells stimulated by TPA for 6 days
also showed the slight activity. By immunocytochemistry,
fluorescence was observed in the cytoplasm of the HL60
cells stimulated by RA or TPA for 4 and 6 days.
TP62
NR3 AND ER-7 NUCLEAR
RECEPTOR SITES OF THE
CYP2B2 PHENOBARBITAL
RESPONSE UNIT:
MUTATIONAL ANALYSIS OF
THEIR IMPORTANCE FOR
PB-DEPENDENT TRANSCRIPTIONAL ACTIVATION
MARIANNE LAIZIER, YANICK PAQUET, and
ALAN ANDERSON
[email protected]
Hepatic CYPs play a critical role in the metabolism of
hydrophobic xenobiotics and certain forms are selectively
inducible. CYP2B1 and CYP2B2 are dramatically induced
by phenobarbital (PB) in rat liver. The CYP2B2 enhancer,
the PB response unit (PBRU), is situated between 2317 and
2155 base pairs upstream of the transcription start site 1,2.
The PBRU contains at least two elements essential for conferring PB responsiveness, NR1 and NR23. They are DR-4
elements located in the PB responsive enhancer module
(PBREM), a 51-base pair sequence within the PBRU3.
When the PBRU or the PBREM is placed directly adjacent
to the basal tk promoter in reporter gene constructs there is
no difference in their ability to confer PB reponsiveness in
cultured primary hepatocytes3,4. However, when both are
tested in the natural sequence context, the PB response
conferred by the PBRU is at least four-fold higher than that
of the PBREM4. These results and others4 suggest the existence of a third sequence element in the PBRU required
for maximal PB responsiveness. Two candidates for this
third element are known, ER-72 and NR35, which are putative nuclear receptor sites that share a half site. Mutation of
their shared half site reduces but does not abolish PB responsiveness, as does mutation of any half site of NR1 or
NR24. To determine which of NR3 or ER-7 are involved in
conferring PB responsiveness, their unique half sites were
mutated within the PBRU. The mutated plasmid reporter
constructs were then tested in the natural sequence context
for their ability to confer PB responsiveness in cultured
primary rat hepatocytes. When the NR3 half site not shared
with ER-7 was mutated, PB responsiveness was reduced
but not abolished, and when either NR3 half site mutation
S150
was combined with a half site mutation of NR1 or NR2, PB
responsiveness was essentially abolished. On the other
hand, mutation of the ER-7 half site not shared with NR3
did not reduce PB responsiveness. Taken together, these results indicate that NR3 is the third element within the
PBRU that is required to confer maximal PB responsiveness. The constitutive androstane receptor (CAR), in the
form of a heterodimer with the retinoid X receptor (RXR),
binds to the retinoic acid ß2 response element (ß-RARE)
and to the NR1, NR2 and NR3 sites of the PBRU and activates NR1-driven transcription of reporter genes in cell lines. CAR-RXR binding to the NR sites is thought to play a
key role in conferring PB responsiveness via binding to
NR16,7. Given that NR3 half sites are identical to those of
NR1 except for a single base pair difference5, it might be
supposed that if CAR-RXR binds to NR1 to activate transcription, it would also bind to NR3. To determine if a high
affinity CAR-RXR site could substitute for NR3, the NR3
site was converted into ß-RARE. The PB responsiveness
conferred by this construct was similar to that conferred by
mutational inactivation of either NR3 half site. Thus, as for
NR14, ß-RARE cannot functionally replace NR3. In conclusion, NR3 is the third element required for conferring
maximal PB responsiveness but the receptor which activates it remains to be discovered.
REFERENCES
263 (1995).
Mol. Pharmacol. 53, 597 (1998).
Biol. Chem. 275, 38427 (2000).
(2001).
6. Honkakoski P., Zelko I., Sueyoshi T., Negishi M.: Mol.
Cell. Biol. 18, 5652 (1998).
TP63
CHARACTERIZATION OF
CYTOCHROME P-450 26 (CYP26)
ENZYMATIC ACTIVITY AND
COMPARISON WITH OTHER
CYPS INVOLVED IN
ALL-TRANS-RETINOIC ACID
(ATRA) METABOLISM
ars possible to consider that CYPs probably play a role in
the fine regulation of the nuclear retinoic acid receptors
activation by modulating the biological properties and the
quantity of ligands available.
REFERENCES :
1. Marill J., Cresteil T., Lanotte M., Chabot G.G.: Mol.
Pharmacol., Vol. 58, 1341 (2000).
2. Marill J., Capron C., Idres N., Chabot G.G.: Biochem.
Pharmacol., Vol 63, 937 (2002).
JULIE MARILL , AUDE PICARD-CLOUTIER
and GUY G. CHABOT
Institut National de la Santé et de la Recherche Médicale
(INSERM U-496), Institut Universitaire d’Hématologie,
Hôpital Saint-Louis, Paris, France. [email protected]
Tumors are characterized by an abnormal cell multiplication accompanied by a loss of certain capacities of cellular differentiation. The anti-proliferative and pro-differentiating activities of retinoids, and in particular those of alltrans-retinoic acid (ATRA), have stimulated research towards their use as chemo-preventive and antitumor drugs.
Human cytochrome P-450s (CYPs) 3A7, 1A1, 2C8, 4A11,
and 2C9 have been identified as the most active CYPs involved in the metabolism of ATRA1 and its isomers2. In addition to these CYPs, the novel CYP26 has been shown to
be inducible by ATRA and to selectively oxidize this molecule to several metabolites. Since no activity comparison
has been carried out so far between the principal CYPs involved in ATRA metabolism, the purpose of this work was
to compare CYP26 activity to the other CYPs. We first
stably transfected the CYP26 gene into HEK293 cells. Of
the various clones that showed ATRA metabolism, clones
293-26-15 and 293-26-2 were selected because they showed the best activity. Microsomes were prepared with cells
from clones 293-26-15, and ATRA (10 µM) metabolism
was carried out using 600 µg of protein/ml in a total volume of 500 µl, for a 1 h incubation period at 37 °C (NADPH,
1 mM). In these conditions, 6 metabolites were formed,
4 of which were identified as the 4-oxo-RA, the 4-OH-RA,
the 18-OH-RA and the 5,6-epoxy-RA. Their relative rate
of formation were as follows: 5.7 ± 4.4 nmol /24 h/mg protein for the 4-oxo-RA; 46.7 ± 6.2 nmol /24 h/mg protein for
the 4-OH-RA; 2.2 ± 0.1 nmol /24 h/mg protein for the 18OH-RA; and, 1.5 ± 0.4 nmol /24 h/mg protein for the 5,6epoxy-RA. Compared to CYP3A7 which was found the
most active until now in the oxidation of ATRA, CYP26
activity was at least 10-fold more active (CYP3A7: 0.6
nmol /24 h/mg protein for the 4-oxo-RA; 1.7 nmol /24
h/mg protein for the 4-OH-RA; 0.03 nmol /24 h/mg protein
for the 18-OH-RA). In addition to the difference in activity,
a qualitative difference was also noted in the formation of
2 novel metabolites formed exclusively by CYP26, and not
by CYPs 3A7, 2C8, or 1A1.
Given the importance of the nature of the ligand to elicit specific biological responses to retinoids, it now appe-
S151
TP64
DISTRIBUTION OF GLUTATHIONE S-TRANSFERASES IN
MULLET (LIZA SALIENS)
ASLI KIRIKBAKAN, and ALAATTIN SEN
Department of Biology, Pamukkale University, 20017 Denizli, Turkey. [email protected]
Glutathione S-transferases (GST) are phase II biotransformation enzymes influenced by exposure to foreign compounds and are proposed as biomarkers of environmental
pollution1,2. They catalyze the conjugation of glutathione to
a great variety of xenobiotic compounds3. The distribution
of GSTs in any tissue or cell type may be one of the determinants of its susceptibility to carcinogenesis4. In this
study, the distribution of GSTs in various leaping mullet
(Liza saliens) tissues has been investigated with the use of
1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid
(EA) as substrates. Figure 1 shows the distribution of GST
activity assessed by CDNB and EA in various leaping mul-
Fig. 1.: Distribution of GST activities in leaping mullet tissues assessed by CDNB and EA
let tissues. The maximum activity was found to be present
in liver followed by kidney, testis and intestine with lower
activities. These results suggested that both total GST and
GST-π isozyme showed similar distribution pattern in mullet tissues. In addition, since GSTs play important role in
protection against various toxic chemicals and environmental pollutants, we have studied the effects of various
metals such as Hg, Zn, Cd, Sb, Cu, Co, Ni, Mn, Fe, K, Li,
Ba, Na, Al and some detergents such as Brij 35, CHAPS,
Cholate, Deoxycholate, Emulgen 913, Lubrol, SDS, Triton
X-100, Tween 20 on mullet liver GSTs activities. In vitro
incubations of fish liver cytosols with various concentrations of these metals and detergents showed that GST metabolism of both CDNB and EA in leaping mullet are sensitive to divalent cations and ionic detergents.
This work is supported in part by Turkish Acedemy of
Sciences. We also thank The Scientific and Technical Research Council of Turkey, TBAG-2058 (101T064).
REFERENCES
1. Brown D.A., Bay S.M., Greenstein, D.J., Szalay P.,
Hershelman G.P., Ward C.F., Westcott A.M., Cross
J.N.: Mar. Environ. Res. 21, 135 (1987).
2. Jimenez B.D., Oikari A., Adams S.M., Hinton D.E.,
McCarthy J.F.: In Biomarkers of environmental contamination (McCarthy J.F., Shugart, L.R. , eds.), Lewis
Publishers, 1990.
3. Melgar Riol M.J., Nóvoa Valiñas M.C., Garcia Fernández M.A., Pérez Lõpez M., Comp. Biochem. and Physio. 128C, 227 (2001).
4. Coles B., Ketterer B.: Biochem. Mol. Biol. 25, 49
(1990).
TP65
BIOCHEMICAL, MOLECULAR
GENETIC AND ENVIRONMENTAL ASPECTS OF MULLET
(LIZA SALIENS) CYP1A1
ALAATTIN SENa, ASLI KIRIKBAKANa,
DONALD R. BUHLERb, AND EMEL ARINCc
a
Department of Biology, Pamukkale University, 20017 Denizli, Turkey, bDepartment of Environmental and Molecular
Toxicology, Oregon State University, Corvallis, Oregon,
97331 USA, cDepartment of Biological Sciences, Middle
East Technical University, 06531 Ankara, Turkey
[email protected],
The CYP1A1 gene encodes microsomal cytochrome
P4501A1 that plays important roles in the metabolism, detoxification and bioactivation of carcinogens and other xenobiotics, including polycyclic aromatic hydrocarbons
(PAH)1. It is one of the most widely studied CYP because it
S152
metabolizes a large number of cytotoxic and/or mutagenic
xenobiotics. Induction of CYP1A1 enhances the metabolism of those chemicals, and therefore, represents an adaptive response of cells to changes in their chemical environments2. We have obtained P4501A1 in a highly purified
form with a specific content of 17 nmol/mg protein from liver microsomes of feral fish, leaping mullet (Liza saliens)
captured from Izmir Bay on the Aegean coast of Turkey3.
Purified mullet CYP1A1 showed very high substrate specificities for 7-ethoxyresorufin and 7-methoxyresorufin in
a reconstituted system containing purified fish P450 reductase and lipid. In addition, EROD activity was strongly inhibited by a-naphthoflavone. Mullet CYP1A1 did not catalyze monooxygenations of other substrates such as aniline,
ethylmorphine, N-nitrosodimethylamine and p-nitrophenol. Antibodies produced against CYP1A1 orthologues in
fish such as trout and scup showed strong cross-reactivity
with the purified mullet CYP1A1. Anti-mullet CYP1A1
antibodies showed very weak cross-reactivity with two
proteins (presumably CYP1A1 and CYP1A2) in 3MC-treated rat liver microsomes. Moreover, 3MC-treated rat liver
microsomal EROD activity was weakly inhibited by the
anti-L. saliens liver CYP1A1. In addition to biochemical
studies with purified mullet CYP1A1, a 2,037 bp CYP1A1
cDNA (GenBank AF072899) was cloned through screening of a cDNA library constructed from leaping mullet
using rainbow trout CYP1A1 cDNA as a probe. This clone
has a 1,563 bp open reading frame (ORF) encoding a 521amino acid protein (58,972 Da)4. Alignment of the deduced
amino acids of CYP1A1 cDNAs showed 58% and 69-96%
identities with human and 12 other fish species, respectively. Southern blot analysis suggested that this CYP1A1
cDNA was from a single copy gene. The leaping mullet
CYP1A1 gene is probably split into 7 exons. Alignment of
the CYP1A1 cDNA encoded amino acids from 13 fish and
7 mammalian species disclosed differences in highly conserved amino acids between aquatic and land vertebrates.
These results strongly suggested that the purified mullet
CYP1A1 is structurally, functionally and immunochemically similar to the CYP1A1 homologues of other teleost
species but functionally and immunochemically distinct
from mammalian CYP1A1. Furthermore, induction of
P4501A1-associated EROD activity of cytochrome
P4501A in leaping mullet were used as biomarker for the
assessment of organic pollutants around the Izmir on the
Aegean Sea coast, Turkey. Fish were captured in February
and May 2002 from four different sites, namely Pasaport,
Foca, Cesme and Mordogan. The results indicated that
these sites are highly contaminated with PAH and/or PCB
type organic pollutants.
This work is supported in part by Turkish Academy of
Sciences.
REFERENCES
1. Lewis D.F. and Pratt J.M.: Drug Metab. Rev. 30, 739
(1998).
2. Whitlock J.P.: Ann. Rev. Pharmacol. Toxicol 39, 103
(1999).
3. Sen A. and Arinc E.: Comp.Biochem.Physiol. 121C,
249 (1998).
4. Sen A., Hu C-H., Urbach E., Wang-Buhler J-L.,YeaHuey Yang Y-H., Arinc E., and Buhler D. R.: J. Biochem. Mol. Toxicol. 15, 243 (2001).
T66
ALBENDAZOLE INDUCED
CYP1A ACTIVITIES IN
MOUFLONS (OVIS MUSIMON)
LENKA SKÁLOVÁa, VENDULA BALIHAROVÁa,
JAKUB VELÍKb, BARBORA SZOTÁKOVÁa,
VLADIMÍR WSÓLa, and JI¤Í LAMKAb
a
Department of Biochemical Sciences, bDepartment of
Pharmacology and Toxicology, Faculty of Pharmacy,
Charles University, Heyrovského 1203, CZ-50005, Hradec
Králové, Czech Republic,[email protected]
Albendazole (ABZ) belongs to anthelmintics long-term
and frequently used in veterinary therapy. In mammals
ABZ undergoes a two-step sulphoxidation resulting firstly
in formation of biologically active ABZ sulphoxide followed by formation of inactive ABZ sulphone. CYP3A (together with FMO) takes part in first oxidation while second
oxidation is mediated by CYP1Ae.g.1. In several species
ABZ and its metabolites cause induction of CYPs, mainly
CYP1Ae.g.2. As the mouflons, food-producing animals used
as farm and game species, are treated with ABZ often, the
effect of ABZ on mouflon biotransformation enzymes was
tested in vivo.
Adult mouflon ewes from game enclosure Vlkov
(Czech republic) were divided in two groups: first group
(5 animals) was treated repeatedly by therapeutic doses of
ABZ (orally individually, in suspension, 5 x 7.5 mg/kg of
body weight), second group (3 animals) represented controls. 24 hours after the termination of experimental treat-
Fig. 1.
S153
ment all animals were culled. Whole liver and proximal
small intestine (1.5 m from stomach) were removed immediately and stored in liquid nitrogen during transport to laboratory. Microsomes were prepared from homogenates of
both tissues of individual animals.
Activities of 7-ethoxyresorufin-O-deethylase (EROD),
7-methoxyresorufin-O-demethylase (MROD) (specific for
CYP1A in rat, human), 6(-testosterone hydroxylase (specific for human CYP3A), 7-methoxy-4-trifluoromethyl-coumarin demethylase (specific for human CYP2C9) and
p-nitrophenol-UDP-glucuronosyl transferase were measured in microsomes.
Significant increase (4-7 times) of CYP1A corresponding activities (EROD and MROD) was found in liver microsomes from ABZ treated animals comparing to control
animals. ABZ treatment also caused significant increase of
EROD activities (6-10 times) in intestinal microsomes (see
Figure). All other activities tested did not significantly differ in ABZ treated and control animals. Increase of
CYP1A proteins by ABZ treatment of mouflon was confirmed by Western blotting. With respect to our results, ABZ
(in dosage scheme used) induces CYP1A in mouflon. Induction of CYP1A could result in increase of ABZ deactivation and could contribute to rise of parasites resistance.
This work was supported by Grant Agency of the
Czech Republic, Grant No. 524/03/1361 and Grant Agency
of Charles University, Grant No.96/2002/C/FAF
REFERENCES
1. Delatour, P. et al. Xenobiotica. 21, 217 (1991).
2. Asteinza, J. et al. Environmental Toxicology and Pharmacology. 9, 31 (2000).
TP67
CYTOCHROME P450 3A4
EXPRESSION IS REDUCED
IN THE PRESENCE OF
CANCER-ASSOCIATED
INFLAMMATION IN
A HUMANISED CYP3A4
TRANSGENIC MOUSE MODEL
KELLIE SLAVIEROa, ANNE LEHNERTb,
SANDIE BIERACHb, CHRIS LIDDLEb,
STEPHEN CLARKEc, LAURENT RIVORYd, and
GRAHAM ROBERTSONb.
a
Department of Pharmacology, University of Sydney, Sydney,
NSW, Australia; bStorr Liver Unit, Department of Clinical
Pharmacology, University of Sydney, NSW, Australia; cDepartment of Medical Oncology, Gloucester House, Sydney
Cancer Centre, Camperdown, Australia; dJohnson & Johnson Research Pty. Ltd. Australian Technology Park, Everleigh, NSW Australia, [email protected]
Cytochrome P450 (CYP) enzyme activity, including
that of CYP3A4, is reduced in the presence of various inflammatory states in man1. Pro-inflammatory cytokines,
such as interleukin 6 (IL-6), have been shown to transcriptionally down-regulate CYP3A42. CYP3A4 activity is also
reduced in cancer patients as compared to healthy volunteers3 and activity declines as a function of the acute-phase
response as measured by serum C-reactive protein in patients with cancer4. It is important to understand, the mechanism of this interaction as reduced CYP3A4 activity may
compromise the use of anti-cancer drugs due to their narrow therapeutic index. Therefore, we sought a suitable animal model for the investigation of the effect of cancer on
the expression of human CYP3A4. Traditional rodent models are not ideal because of significant species differences
in the activity and regulation of CYP3A genes. Instead,
a tumour-bearing transgenic mouse model was developed.
Briefly, suspensions of Engelbreth-Holm-Swarm sarcoma
cells were injected i.m. into male mice harbouring a transgene construct comprising the 13kb upstream regulatory
region of the CYP3A4 gene linked to the lacZ reporter
gene. This transgenic model has been validated using
known inducers of the CYP3A4 gene. Expression of the
CYP3A4/lacZ transgene was measured by both X-gal staining of liver slices and assay of β-galactosidase activity in
liver extracts. The expression of a mouse CYP3A4 homolog, Cyp3a11, was measured by real-time PCR following
reverse transcription of hepatic mRNA. Serum IL-6 and
TNFα levels were measured by commercially available
ELISA kits. Comparison between control and tumour animals was performed using Mann-Whitney U test and p =
0.05 set for significance. Preliminary results show that tumour-bearing mice (tumour weight approximately 10%
whole body weight) have reduced CYP3A4/lacZ transgene
S154
expression as determined by X-gal staining of liver wedges. β-galactosidase activity was reduced by 50% in tumour-bearing animals compared to saline-treated controls
(p = 0.025). Furthermore, there was a corresponding 60%
repression of mouse Cyp3a11 gene expression in the presence of the tumour as compared to controls (p = 0.014). In
addition, serum IL-6 concentrations were elevated from
undetectable levels in controls to 25.45 +/- 3.76 pg/ml in
tumour-bearing mice compared to controls (0 +/- 0 pg/mL
vs 25.45 +/- 3.76 pg/mL [median +/- SEM], p = 0.002). In
contrast, all animals had serum TNF_ concentrations below
the limit of assay detection (23 pg/mL). Further experiments investigating the time course of the reduction in
CYP3A4/11 activity are currently underway. The implications of these results suggest that the pharmacokinetics,
and hence the pharmacodynamics, of the majority of drugs
used to treat malignancies could be altered in the presence
of cancer and associated inflammation.
REFERENCES
1. Morgan ET: Drug Metab Rev 29, 1129 (1997).
2. Pascussi JM, et al: Biochem Biophys Res Commun 274,
707 (2000).
3. Slaviero KA, et al: Lancet Oncology In press (2003)
4. Rivory LP, et al: British J. Cancer 87, 277 (2002).
TP68
ACTIVITY OF CYTOCHROMES
P450 2C IN RAT
CARDIOMYOCYTES
LUCIE KOUSALOVÁa, JANA BARANOVÁa,b,
JITKA PSOTOVÁc, EVA ANZENBACHEROVÁc, and
PAVEL ANZENBACHERa
a
Institute of Pharmacology, Faculty of Medicine, Palacky
University, Hnûvotínská 3, CZ-775 15 Olomouc,Czech Republic, bSlovak Technical University, Bratislava, Slovakia,
c
Institute of Medical Chemistry and Biochemistry, Faculty
of Medicine, Palacky University, Hnûvotínská 3, CZ-775
15 Olomouc,Czech Republic, [email protected]
The function of cytochromes P450 in the heart is speculated to be in biosynthesis of mediator molecules from endogenous precursors1,2. We focused on the presence and activity
of enzymes of the CYP2C subfamily, which are known to be
involved in formation of derivatives of arachidonic acid2.
The cardiomyocytes of the newborn rat were prepared
by trypsin digestion; the cells were either cultured or used
in a suspension of freshly isolated cells. The presence of
CYP2C enzymes was studied by checking their enzyme
activity (hydroxylation of a prototypical substrate, diclofenac). The results show that the activity of the CYP2C is
clearly present both in the freshly prepared cells as well as
in the samples from cultured cardiomyocytes.
Acknowledgment
The authors (L.K., J.B.) wish to thank the Medical Faculty, Palacky University for the grant support (grants
12101105, 12101107).
TP70
APPLICATION OF
α-NAPHTHOFLAVONE DURING
THE GROWTH AND
DEVELOPMENT OF BROILERS
REFERENCES
1. Fisslthaler B., Michaelis R.U., Randriamboavonjy V.,
Busse R., Fleming I.: BBA 1619, 332 (2003)
2. Thum T., Borlak J.: Br. J. Pharmacol. 130, 1745 (2000).
TP69
METABOLISM OF
α-NAPHTHOFLAVONE AND
ITS THIO-ANALOGUE BY
MICROSOMAL SYSTEM
PETRA VALENTOVÁ, PETR HODEK,
LUCIE BO¤EK-DOHALSKÁ, MIROSLAV ·ULC, and
MARIE STIBOROVÁ
[email protected]
Synthetic and naturally occurring flavonoids are effective inhibitors of several CYPs, e.g. CYP1A1, 1A2, 1B1,
3A4 and CYP19. In addition, certain metabolic activities of
CYP3A4 and 1A2 were also stimulated by some compounds of flavonoid structure. Synthetic 7,8-benzoflavone
(ANF) is an inhibitor of human CYP19, CYP1A1 and 1A2,
but an activator of CYP3A4. Moreover, a newly synthesized thio-analogue of ANF (containing sulphur in flavonoid
skeleton instead of C4 oxo-group) (SANF) is also an activator of CYP19. Modulation of CYP activities by flavonoids has been extensively studied because of their potential
use as agents blocking the initiation stage of carcinogenesis.
However, much less is known about their metabolism. Microsomes of rifampicine-treated rabbit were used as a model
system to study of ANF and SANF metabolism. They contain high levels of CYP3A6 which is expected to be the major CYP present in intestinals and is in contact with flavonoids ingested. Using RP-HPLC combined with MALDITOF, 5,6-dihydrodiol, 7,8-dihydrodiol and 5,6-epoxid of
ANF were determined as the major ANF metabolites. On
the other hand, SANF is not metabolized into any hydroxyderivatives in the microsomal system used. This compound
is converted to ANF first and then ANF formed in this reaction undergoes its regular metabolism. Role of other
CYPs in the metabolic conversion of both flavonoids is underway in our laboratory.
S155
ALENA MIâÁKOVÁa, PETR HODEKb,
MARTIN POPL·TEINa, and PAVEL TREFILa
a
BIOPHARM, Research Institute of Biopharmacy and Veterinary Drugs, a.s., Jílové near Prague, The Czech Republic, [email protected], bDepartment of Biochemistry, Charles
University in Prague, Hlavova 2030, Prague 2, Czech Republic, [email protected]
The modulation of CYP19 (aromatase) activity might
significantly change the estrone/testosterone ratio and consequently change the poultry sex. Inhibition of CYP19 activity or expression causes a serious hormonal status
change. For example, complete ovariectomy of female
chicken results in a phenotypic sex-reversal accompanied
by the development of testoids1. Similarly, in ovo administration of Fadrazole (CYP19 inhibitor), elicited in the case
of genotypic females, testes formation. In the late development of the treated animals, however, the sex is frequently
reversed to the genetic one, including the development of
related sex organs2. Hence, it is clear, that after a single treatment of embryos, inhibitors have to be applied repeatedly
to the hatched chicken to maintain the established sex reversal. Since the CYP19 was also detected in males, namely in testes, administration of CYP19 inhibitors might
be expected to change hormone status of cocks with the
respect to enhanced testosterone production on the account
of a blocked androstenedione conversion to estrone.
α-naphthoflavone seems to be the best model inhibitor of
CYP19 for naturally occurring flavonoids3. In our experiments we verified the influence of repeated postnatal oral
administration of α-naphthoflavone to broilers on the
growth rate and possible physiology changes of their body.
Application of α-naphthoflavone within 42 days of the fattening period of broiler chickens resulted in statistically
significant difference (P<0.01) in the weight of the testes
(4.7g) in comparison to controls (1.2g). We did not find
statistic difference between the size of the left and right
testes. Moreover, spermatogenesis in the group where
α-naphthoflavone was applied began very early - the 10 th
week and cocks in this group started to crew at 13 weeks of
age, at least 4 weeks earlier than control group. Ovary in
experimental group treated with α-naphthoflavone was heavier (P<0.05) in comparison to control groups. The beginning of laying period in the (-naphthoflavone treated hens
started at least one week earlier comparing to the control.
For male groups, there was no difference in livers weight
between treated and untreated animals. In contrary, female
groups, showed significant increase (P<0.05) in liver weight in experimental group treated with (-naphthoflavone.
There was no statistically significant difference between
the growth rate of treated and untreated male or female
chickens. By the carcass analysis of the live body weight,
kidney, gizzard, heart, abdominal fat and breast muscle weight we also did not find any differences from control
groups.
The work was supported by grants GACR 523/01/0840 from Grant Agency of the Czech Republic
and MSM-113100001 from The Czech Ministry of Education.
REFERENCES
1. Wallenburg, J.: Gegenbaurs Morphol. Jahrb. 128, 463
(1982).
2. Burke, W.H., Henry, M.H.: Poultry Sci. 78, 1019
(1999).
3. Hodek, P., Trefil, P., Stiborová, M.: Chem. Biol.
Interact. 139, 1 1 (2002).
S156
05 PostWP.QXD 10.6.2003 11:57 Stránka 157
7
Poster Presentations – WP
POSTERS – WEDNESDAY – WP
WP02 AFLATOXIN B1 BINDING TO
MUTANT PHE209ALA CYP2A5
EXPRESSING SACCHAROMYCES CEREVISAE YEAST CELLS
WP01 MOLECULAR BREEDING OF
CYTOCHROME P450 ENZYMES
INVOLVED IN XENOBIOTIC
METABOLISM
PETRA BARTAKOVAa, HANI EL-NEZAMIB, HANNU
RAUNIOa, and RISTO O. JUVONENa
a
Departments of Pharmacology and toxicology, and
Clinical Nutrition, University of Kuopio, POB 1627,
70211 Kuopio, Finland
NEDELJKA ROSICa, THIERRY G.A. LONHIENNEa,
JAMES J. DEVOSSb, and ELIZABETH M.J. GILLAMa
b
a
Department of Physiology and Pharmacology, School of
Biomedical Sciences, and bDepartment of Chemistry,
School of Molecular and Microbial Sciences, The University of Queensland, St. Lucia, Australia, 4072
[email protected]
DNA shuffling, a novel technique for molecular evolution in vitro, can be used in designing new enzymes with
improved properties. Xenobiotic-metabolizing P450s are
especially well suited as starting materials for creating better catalysts because of their wide substrate specificity. In
the present study, a novel method for DNA shuffling was
developed using appropriate restriction enzymes in the initial digestion step, followed by size-selective filtration for
selection fragments under 300bp. P450s 2C9, 2C11 and
2C19 were subjected to a single round of shuffling and
co-expressed with NADPH-cytochrome P450 reductase in
E. coli. A sample (54 clones) of the resultant library was
assessed for sequence diversity, hemo- and apoprotein expression, and activity towards the substrate indole. All mutants showed restriction fragment length polymorphism
patterns that were different when compared to all parents,
suggesting that the library was free from contamination by
any parental forms. Preliminary sequencing of mutants
confirmed that significant mixing of different P450s had
been achieved. Significant hemoprotein expression was detectable in 27/54 (50%) of mutants and in a further 13/54
(24%) of sampled mutants, expression was marginal. Indigo production was less than or comparable to the activities of the parental P450s, but certain mutants showed
a shift towards indirubin production. In conclusion, a method is presented for effective shuffling of P450 sequences
to generate libraries of novel mutant P450s. The library generated using P450s 2C9, 2C11 and 2C19 showed significant diversity and was free from contamination by parental
forms.
S157
Introduction
Carcinogenic aflatoxin B1 (AFB1) is metabolized to the
AFB1 exo-8,9-epoxide which is a primary metabolite responsible for its toxic effects1. This metabolite is conjugated
to inactive glutathione derivative, although it reacts also
with DNA forming stable adducts or is hydrolyzed to
AFB1 dihydrodiol. Dihydrodiol is in equilibrium with dialdehyde, which can react with protein or be reduced to monoaldehydes. CYP enzymes and CYP2A5 in mice catalyze
the formation of AFB1 8,9-epoxide. When CYP2A5 or its
mutants are expressed constitutively in S. Cerevisiae yeast
cells and simultaneously exposed to AFB1, the viability of
the cells is decreased corresponding with the ability of
CYP mutant to produce AFB1 8,9-epoxide2. Especially the
mutant Phe209Ala CYP2A5 efficiently bioactivates AFB1.
This study aimed at investigating (1) the effect of AFB1
concentration and exposure time on the viability of
Phe209Ala CYP2A5 mutant expressing yeast cells (mutant
CYP2A5) and (2) the binding of AFB1 to these cells. To
study the effect of time and concentration, CYP2A5 mutant
yeast cells were incubated for 5 - 80 min with liquid growing media containing 0.25, 1 or 4 µM AFB1. Binding of
AFB1 was studied by incubating yeast cells with tritium labelled AFB1 and the radioactivity in the yeast cells indicated its binding. In both studies, yeast cells expressing no
foreign CYP enzyme (pAAH5) were used as a reference.
Results
The viability of mutant CYP2A5 yeast cells was dependent on the incubation time and concentration of AFB 1.
At 0.25 µM AFB1, after 5 min the viability was 80 %, this
was decreased to 40 % at 80 min. At 4 µM the viability was
decreased to 20 % already at 5 min, however, no further reduction in the viability was observed by prolonged exposure time. Such findings were not observed with pAAH5
yeast cells. When mutant CYP2A5 yeast cells were incubated with tritium labelled AFB1, radioactivity detected in
yeast cells was significantly high and was dependent on the
amount of yeast cells, concentration of AFB1 and incubation time. Moreover, washing the yeast cells with methanol
could not remove this radioactivity. This was not the case
for the pAAH5 yeast cells, where the radioactivity was
small and was not dependent on variables mentioned
above.
Conclusions
Mutant CYP2A5 in yeast cells metabolize AFB1 to its
reactive epoxide and due to this metabolism it is bound covalently to yeast cells resulting lowered viability of these
yeast cells. Mutant CYP2A5 expressing yeast cells are
a good model to study the toxicity of AFB1.
REFERENCES
1. Guengerich F.P., Johson W.W.: Drug Metab. Rev. 31,
141 (1999)
2. Pelkonen P., Lang M.A., Negishi M., Wild C.P., Juvonen R.: Chem. Res. Tox. 10, 85, (1997)
WP04 VALIDATION OF COMMERCIALLY SUPPLIED CRYOPRESERVED HEPATOCYTES FROM
A RANGE OF SPECIES FOR USE
IN METABOLIC PROFILING
STUDIES
CLIVE DILWORTH,
REBECCA SHANNLY-HENDERSON,
VICTORIA EAGLING, PETER D FENN,
and GRAHAM R MURRAY
DMPK Department, Quintiles Limited, Research Avenue
South, Heriot Watt University Research Park, Riccarton,
Edinburgh, EH14 4AP, UK, [email protected]
WP03 ENGINEERING SUBSTRATE
RECOGNITION IN CATALYSIS
BY CYTOCHROME P450CAM
STEPHEN G. BELLa, XUEHUI CHENb,
FENG XUb, ZIHE RAOb, and LUET-LOK WONGa
a
Department of Chemistry, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1
3QR, U.K., bLaboratory of Structural Biology, School of
Life Sciences and Engineering, Tsinghua University, Beijing 100084, China.
Abstract
We have a continuing interest in applying the current
knowledge of P450cam substrate recognition to engineer
the enzyme for the biotransformation of unnatural substrates with the long-term aim of applications in the synthesis
of fine chemicals and bioremediation of environmental
contaminants. Comparison of the structure of target substrates with camphor, the natural substrate, lead to the design of active site mutations that had greatly enhanced
activity for the oxidation of chlorinated benzenes and selectivity of (+)-α-pinene oxidation. The crystal structure of
the F87W/Y96F/V247L mutant with 1,3,5-trichlorobenzene and (+)-α-pinene bound revealed the enzyme substrate contacts and provided insights into the activity and
selectivity patterns. The structures also provided a novel
basis for further engineering of P450cam for increased activity and selectivity for the oxidation of related compounds.
S158
Isolated hepatocytes are a well established cellular model providing metabolic information on new molecular entities (NME). Traditionally, metabolic studies have used
microsomes, however the main advantage of isolated hepatocytes is that they contain the cofactors and drug-metabolising enzymes required to study integrated Phase I and
Phase II metabolism reactions. The use of cryopreserved
hepatocytes has increased in recent years due to a number
of reasons namely, the lack of human tissue available, a desire to decrease animal usage and improvements in the cryopreservation process.
We have validated a model for studying the metabolic
profiles of NMEs utilising commercially available cryopreserved hepatocytes from a range of species. As human
hepatocytes are the „gold standard“ to which metabolic information from other species is compared, freshly isolated
human hepatocytes were also investigated for comparative
reasons. Cryopreserved rat, rabbit, dog, primate and human
hepatocytes were obtained from Xenotech, (KS, USA) or
In Vitro Technologies (MD, USA). Freshly isolated human
hepatocytes were supplied by the UK Human Tissue Bank.
Following thawing of the cryopreserved hepatocytes,
the viability and the metabolic activity of the cells were determined over a 3 hour incubation period. Cell viability
was assessed using the Trypan exclusion test. Metabolic
activity of the hepatocytes was measured using testosterone 6β-hydroxylation as an marker of Phase I metabolism.
Phase II metabolism was assessed by measuring the glucuronidation and sulphation of the probe substrate 4-methylumbelliferone. In all species, following thawing, Phase
I and II metabolic activities were present. Furthermore
these activities were demonstrated to be maintained
throughout the 3 hour incubation period. There was only
a small decrease in cell viability observed over the 3 hour
incubation period. Therefor, this data indicates that commercially obtained cryopreserved hepatocytes are suitable
for in vitro metabolic profiling studies with NMEs.
WP05 P450 2E1/BMR: STUDIES OF
A HUMAN/BACTERIAL P450
FUSION PROTEIN
WP06 O2 METABOLISM BY SOLUBLE
CYTOCHROME P450 REDUCTASE AND ITS CHIMERAS WITH
C-TERMINI OF NITRIC OXIDE
SYNTHASES ASSAYED BY DIACETYLDEUTEROHEME HRP
MICHAEL JAMES FAIRHEAD, and
GIANFRANCO GILARDI
Imperial College London, Department of Biological Sciences, South Kensington, London SW7 2AZ, [email protected]
The cytochrome P450s form a large family of monooxygenase enzymes that carry out an essential role in xenobiotic metabolism1. In humans they are found on the membranes of the smooth endoplasmic reticulum where they
are attached by a N-terminal anchor2. Human P450s can be
expressed in non-native hosts such as Escherichia coli in
a membrane bound form but for optimal activity need to be
fused to or co-expressed with the cytochrome P450 reductase3. To overcome these limitations we have made a chimerical protein containing the N-terminal of modified human P450 2E14 fused to the soluble bacterial cytochrome
P450 reductase from Bacillus megaterium (BMR). The
construct, P450 2E1/BMR, consists of amino acids 1-472
from modified P450 2E1 and amino acids 473-1049 from
BMR. The fusion protein has been expressed using the
pCW plasmid vector in the DH5α strain of Escherichia coli
at a level of 900 nmol of P450 per litre of culture. The protein has been purified in a holo form and demonstrates pnitrophenol and chlorzoxazone hydroxylase activities, both
marker activities for P450 2E15,6. The protein also shows
a P450 peak upon carbon monoxide binding, characteristic
of an active protein and exhibits spin shifts on substrate
binding. In summary these results indicate that the construct retains the marker activities of human P450 2E1, i.e.
the ability to metabolise p-nitrophenol and chlorzoxazone,
but with the added advantage of being catalytically selfsufficient and soluble for electrochemical applications7.
The P450 2E1/BMR construct should prove useful in studies of the human P450 2E1 enzyme in a convenient format in both whole Escherichia coli cells and as a purified
enzyme.
REFERENCES
1. Nebert D.W., Russel D.W., Lancet 360, 1155 (2002).
2. Black S.D., FASEB J. 6, 680 (1992).
3. Gonzalea F.J., Korzekwa K.R., Annu. Rev. Pharmacol.
Toxicol. 35, 369 (1995).
4. Gillam E.J.,Guo Z., Guengerich F.P., Arch. Biochem.
Biophys. 312, 59 (1994).
5. Koop, D.R., Mol. Pharmacol. 29, 399 (1986).
6. Peter R., Bocker R., Beaune P.H., Iwasaki M., Guengerich F.P., Yang C.S. Chem. Res. Toxicol. 3, 566 (1990).
7. Gilardi, G., Meharenna Y.T., Tsotsou G.E., Sedeghi
S.J., Fairhead M.J., Giannini S., Biosens. Bioelec. 17,
133 (2002).
S159
YUZURU ISHIMURAa, SATYA P. PANDAa,
MARIE JÁCHYMOVÁa,b, PAVEL MARTÁSEKb,
TATSUYA KITAZUMEc, JULIAN A. PETERSONc,
LINDA J. ROMANa, and BETTIE SUE S. MASTERSa.
a
Dept.of Biochem., Univ. of Texas Health Sci. Ctr. at San
Antonio, TX 78229-3900, bThe Inst. of Clin. Biochem. and
Dept. of Pediatrics, Faculty Gen’l Hosp. & Charles Univ.,
Prague 2, Czech Republic and cDept. of Biochem., Univ. of
Texas Southwestern Med. Ctr., Dallas, TX 75235-9038,
USA, [email protected]
Diacetyldeuteroheme-substituted horseradish peroxidase
(dHRP) reacts with both superoxide anion (O2-) and hydrogen
peroxide (H2O2) to form its compounds III and II, respectively. These compounds are rather stable and spectrally distinguishable from each other. Hence it has been used to detect and quantify the oxygen metabolites of some oxidases1.
Recently, we constructed chimeras of a soluble CYPOR and
C-terminal tail portions of neuronal, endothelial and inducible NOS’s, and have elucidated their kinetic properties2. In
the present study, we have applied the dHRP method to analyze oxygen metabolites of these chimeric enzymes of CYPOR in their reactions with NADPH under aerobic conditions. The most common method to detect and quantify O2- based on SOD-sensitive cytochrome c reduction is difficult to
apply to these reactions, because CYPOR reduces cytochrome c directly, not via O2-. Results obtained in combination with the measurement of O2 consumption using an oxygen monitor indicate that the soluble CYPOR reduces O2
exclusively to O2- and not to H2O2. The findings confirm our
previous results using the adrenochrome method on steapsinsolubilized CYPOR3, indicating that CYPOR produces only
O2-. Available evidence suggests that the CYPOR chimeras
with NOS tails produce both O2- and H2O2. Experiments using
the dHRP method to analyze oxygen metabolites from wild
type and chimeric constructs of holoNOS are in progress.
Supported by NIH Grants GM52419 and HL30050 and
Grant No. AQ-1192 from the Robert A.Welch Foundation
to BSSM. PM was supported by Grant GAUK
10/2002/c from the Czech Republic.
REFERENCES
1. Makino, R. et al.: J. Biol.Chem. 261, 11444 (1986).
2. Jáchymová, M. et al.: Nitric oxide 6, 404(2002).
3. Prough, RA, Masters, BSS: Ann. NY Acad. Sci. 212, 89
(1973).
SPA is a homogeneous technology that has been used
extensively within the pharmaceutical industry. It has been
used in a broad range of applications including radioimmunoassays, receptor-ligand and enzyme assays.
The assay that will be described measures the conversion of the CYP2D6 substrate, [14C]dextromethorphan to
[14C]formaldehyde, by selective capture of this reaction
product using modified Yttrium silicate SPA beads4.
Validation of the assay against a panel of known
CYP2D6 inhibitors has been carried out and is summarised
in Table1.
WP07 AN HOMOGENEOUS ASSAY
FOR SCREENING CYP2D6
INHIBITORS USING
SCINTILLATION PROXIMITY
ASSAY TECHNOLOGY
EMILY BEYNON, MOLLY PRICE-JONES,
JENNY BERRY, and KELVIN T. HUGHES
Amersham Biosciences UK, Limited, The Maynard Centre,
Forest Farm, Whitchurch, Cardiff, UK, CF14 7EG;
The P450 cytochromes (CYP) are a superfamily of enzymes found in the liver which catalyse the oxidative conversion of drugs and other lipophilic compounds to hydrophilic metabolites. This conversion allows for the elimination or further metabolism and elimination of the drug from
the body. CYP2D6 and CYP3A4, izoenzymes of this family, catalyse the metabolism of the major proportion of
drugs available on the market.
Inhibition of CYP-mediated metabolism accounts for
a number of adverse drug-drug interactions1, which often
leads to withdrawal from the market2,3. Promising new chemical entities (NCEs) are routinely tested for CYP inhibition during drug discovery or development.
A high throughput Scintillation Proximity Assay (SPA)
to identify compounds that inhibit P450 CYP2D6 has been
developed.
Tab. 1. Results obtained with a panel of test compounds.
SPA
95% confidence
intervals
Diazepam
Determined not to inhibit CYP2D6 in the
Verapamil
SPA assay and in literature.
Literature
Omeprazole
295
256-342
240.7
Tranylcypromine
40.2
35.1
28.1-43.9
33.2-48.6
30
Tropisetron
16.5
16.0
12.6-21.6
1.3-21.0
14
Desipramine
3.7
4.1
3.1 - 4.3
3.8 - 4.4
12.5
Loratadine
1.99
1.75 - 2.27
2.7
Sulconazole
0.35
0.29
0.25 - 0.49
0.24 - 0.36
0.4
Quinidine
0.0054
0.0077
0.0058-0.010
0.0050-0.0061
0.018 0.043
Lansoprazole
7.0
8.2
6.3-7.7
7.5-9.1
44.7
Metoclopromide
23.1
18.2
17.5 - 30.5
13.4 - 24.9
4.7
1. Guengerich F.P.: Drug-Drug Interactions; Scientific and
Regulatory Perspectives. San Diego: Academic Press,
pp. 7-35 (1997).
2. Peek C.C., Temple R., Collins J.M.: JAMA, 269, 1550,
(1993).
3. Po A.L.W., Zhang W.Y.: Lancet, 351, 1829,(1998).
4. Delaporte E., et al., J. Biomolecular Screening, 6, 225,
(2001).
WP08 EXPLORATION OF NATURAL
AND ARTIFICIAL P450
SEQUENCE SPACES
P. URBAN, V. ABÉCASSIS, G. TRUAN,
and D. POMPON
Centre de Génétique Moléculaire CNRS UPR2167,
91190 Gif-sur-Yvette, France
ki values µM
Compound
REFERENCES
Two complementary methods are described that associate in vitro and in vivo steps to generate P450 sequence diversity by segment directed mutagenesis and family shuffling. High-throughput DNA chip based and HPLC technologies have been developed for the characterization of
combinatorial libraries and for the characterization of activities shuffling achieved, respectively.
Using these approaches, two combinatorial libraries of
variants derived from the human CYP1A subfamily were
constructed and their sequence diversity characterized. The
results of functional screenings using high-throughput tools for the characterization of membrane P450-catalyzed
activities is presented with a preliminary discussion.In
contrast to mutants CYP1A1 in the 204-214 segment,
CYP1A1-CYP1A2 mosaic structures with, on average,
5 crossovers exhibit a wide range of substrate selectivities.
These mosaic structures also discriminate between closely
related alkoxyresorufin and polycyclic aromatic hydrocarbon substrates. These results open the way to global highthroughput analysis of structure-function relationships in
eukaryotic P450s using combinatorial libraries of enzymes
together with a library of structurally related substrates.
S160
This poster illustrates how in vitro and yeast in vivo
steps can be associated to improve the diversity of combinatorial libraries either resulting from segment-directed
mutagenesis or from family shuffling. The CLERY familyshuffling procedure combines an in vitro DNA-suffling
PCR step to an in vivo recombination step in yeast aims to
build a library of shuffled sequences containing a low proportion of parental structures. The recombination step in
yeast is both a self-cloning tool and an additional shuffling
step when overlapping and partial-length PCR-shuffled
DNAs are involved. The production of such segment-limited or family-shuffled combinatorial libraries in yeast specially engineered to enhance P450-catalyzed activities is of
great interest for the functional analysis of membranebound shuffled P450 variants.
WP09 CYTOCHROME P4502E1
ACTIVITY AND FREE RADICAL
OXIDATION IN THE LIVER
OF HYPOTHYROID RATS
WITH ACUTE ALCOHOL
INTOXICATION
the liver cytosolic fraction from hypothyroid ethanol-fed
rats the activity of alcohol dehydrogenase was increased.
A decrease in MDA levels (28 and 56%) and an increase in
glutathione content (22 and 102%), as well as an activation
of glutathione peroxidase and glutathione reductase were
found. A decrease in CYP2E1 and CYP-reductase activities in hypothyroidism can lead to a reduction in microsomal oxygen consumption and free radical production, thereby limiting ethanol-induced oxidative stress.
WP10 ASSIGNMENT OF OMEPRAZOLE-SENSITIVITY GENE LOCUS,
WHOSE PRODUCT MEDIATES
CYP1A1 INDUCTION, ON
HUMAN CHROMOSOME 10
HIDEAKI KIKUCHIa, SHINICHI FUKUSHIGEb,
MASAHIKO SHIBAZAKIa, AHMED SOHELa, and
TAKASHI TAKEUCHIa
a
Department of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryomachi, Aoba-ku, Sendai, hkikuchi@idac,tohoku.ac.jp, bDepartment of Molecular Pathology, Tohoku University
School of Medicine, 4-1 Seiryo-machi, Aoba-ku, Sendai
NATALLIA KALOSHYNA, TAMARA KHOMICH, and
PAVEL PRONKO
Institute of Biochemistry NASc,230017,BLK-50 , Grodno,
Belarus, [email protected]
Ethanol-inducible cytochrome P450 2E1 (CYP2E1) is
involved in free radical generation during ethanol oxidation by liver microsomes. A complicated relationship
exists between the ethanol effects on the thyroid function
and the thyroid hormone influence on the metabolism and
toxicity of ethanol. The aim of our research was to study liver CYP2E1 activity, lipid peroxidation rate and antioxidant defence in acute alcohol intoxication of rats with hypothyroidism. The experimental hypothyroidism in rats
was provoked by treatment with mercazolil (5 and 20
mg/kg, for 14 d.), ethanol was administered singly (6 g/kg,
i.g.).The developtment of hypothyroidism was followed by
measuring plasma thyroid hormone (T3 and T4) levels. In
liver microsomes, CYP2E1 activity was 35% lower in the
hypothyroid rats as compared to the control animals,
whereas any differences in cytochrome P450-reductase
(CYP-reductase) activity were not found in these groups.
The ethanol administration caused an activation of
CYP2E1 (23%) and CYP-reductase (19%) in euthyroid rats
and a significant increase in CYP2E1 activity in liver microsomes of mercazolil-treated rats (5mg/kg).The group
with more pronounced hypothyroidism showed no alcohol
effect on CYP2E1 activity. The CYP-reductase activity
was decreased proportionally to the reduction of T 3 and T4
levels in the blood after the acute ethanol administration. In
S161
Previously, we reported that CYP1A1 was induced in
human GepG2 cells, but not in mouse Hepa-1 cells, by
such benzimidazole compounds as omeprazole (OP), lansoprazole, and thiabendazole1,2. We also showed that protein tyrosine kinase is involved in the induction of
CYP1A1 by omeprazole treatment in human HepG2 cells,
and that the signal transduction pathway for omeprazole is
different from that existing TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin). Furthermore, we reported that in HepG2
cells, the Ah-receptor antagonist (3’-methoxy-4’-aminoflavone) blocked the induction of CYP1A1 by TCDD, but not
produced by OP3. Consequently, we proposed the presence
of a ligand-independent pathway for the activation of the
Ah receptor, and the omeprazole-sensitivity gene product
may be involved in this pathway.
We utilized the lack of CYP1A1 induction by OP in
Hepa-1 cells to map a putative human gene for OP-responsiveness in cell hybrids produced by fusion of Hepa-1 and
HepG2 cells. OP-induced CYP1A1 expression was detected in four out of 32 Hepa-1 x HepG2 cell hybrid analyzed.
To help identify the gene locus, a radiation-hybrid cell
(E11) was constructed. Use of reverse-fluorescence in situ
hybridization revealed that these five cell lines commonly
retained human chromosome 10p4.
To narrow down the gene locus of OP-sensitivity gene
on chromosome 10, we used site specific PCR primers and
identified DNA sequences that are commonly present in
the four OP-responsive hybrid cells and radiation-hybrid
cell. Finally, we assigned the two regions (2.1 and 3.0 magabp) that are responsible for the induction of CYP1A1 by OP
treatment on the short arm of human chromosome 10.
REFERENCES
1. Kikuchi H., Hossain A., Sagami I., Ikawa S. Watanabe
M.: Arch. Biochem. Biophys. 316, 649 (1995).
2. Kikuchi H., Kato H., Mizuno M., Hossain A., Ikawa S.,
Miyazaki J. Watanabe M.: Arch. Biochem. Biophys.
334, 235 (1996).
3. Kikuchi H., Hossain A., Yoshida H. Kobayashi S.:
Arch. Biochem. Biophys. 358, 351 (1998).
4. Kikuchi H., Fukushige S., Shibazaki M. Shiratori Y.:
Cytogenet. Genome. Res. 97, 51 (2002).
WP11 STUDY ON ORGAN SPECIFICITY OF CYP1A1 INDUCTION BY
1-PHENYLAZO-2-HYDROXYNAPHTHALENE (SUDAN I) IN
RATS
VLADIMÍRA MARKOVÁ, VÁCLAV MARTÍNEK,
PETR HODEK, and MARIE STIBOROVÁ
various organs has not yet been studied in details. Here, we
study an organ specificity of Sudan I as an inducer of
CYP1A1 in rats. Selective antibodies against rat CYP1A1
were utilized for this study. An expression of CYP1A1 protein was found to be strongly induced in rat liver, kidney,
lung, spleen and heart by treatment of animals with Sudan
I, while almost no CYP1A1 induction was detectable in
brain. The expression of this enzyme was the highest in liver of treated rats (almost one order of magnitude higher
than those in control animals). Analogously, the increase in
CYP1A1 expression correlates with an increase of EROD
activity, a marker for CYP1A1/2, or with that of Sudan
I own oxidation. In addition to CYP1A1, Sudan I induces
DT-diaphorase, the cytosolic enzyme, whose gene(s)
is(are) also transcriptionally activated by foreign chemicals
through the Ah-dependent mechanism. Its induction was
detectable in most rat tissues tested in the study except of
brain. No increase of DT-diaphorase activity by Sudan
I was detectable in rat brain cytosolic samples. The significance of CYP1A1 induction by Sudan I for an increase in
risk of Sudan I exposition for humans working in dye
industry is discussed.
Supported by Grant Agency of Charles University
(241/2001/B/CH/PrF) and Ministry of Education of the
Czech Republic (MSM 113100001).
REFERENCES
[email protected], [email protected],
[email protected]
Sudan I [1-(phenylazo)-2-naphthol, C.I. Solvent Yellow 14] was used as a food coloring in several countries,
but it has been recommended as unsafe, because it causes
tumors in the liver or urinary bladder in rats, mice, and rabbits, and is considered possible carcinogenic and mutagenic for man1,2. Besides its carcinogenicity, Sudan I is a potent contact allergen and sensitizer, eliciting pigmented
contact dermatitis in human.
We found that this carcinogen is metabolized (activated
and/or detoxicated) almost exclusively by CYP1A1; other
CYPs oxidize Sudan I with efficiencies more than one order of magnitude lower than CYP1A11,2. Therefore, Sudan
I oxidation seems to be utilized as a marker for the
CYP1A1 activity. While CYP1A1 is not constitutively expressed in human livers, it seems to be induced by planar
aromatic compounds binding to the aryl hydrocarbon receptor (AhR), e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin
and/or by polycyclic hydrocarbons present in cigarette
smoke3. The CYP1A1 enzyme is even strongly induced by
Sudan I itself in rat livers by this mechanism4. The AhR is
ubiquitously expressed in most organs and cells in the
body. Nevertheless, the de novo synthesis of CYP1A1 molecules as a result of an increased transcription of CYP1A1
gene after stimulation by Sudan I interaction with AhR in
S162
1. Stiborová M., Martínek V., R˘dlová H., Hodek P., Frei E.:
Cancer Res. 62, 5678 (2002).
2. Martínek V., Stiborová M.: Collect. Czech. Chem.
Commun. 67, 1883 (2002).
3. Drahushuk A. T., McGarrigle, B. P., Larsen, K. E., Stegeman, J. J., Olson, J. R.: Carcinogenesis 19, 1361
(1998).
4. Lubet R. A., Connolly G., Kouri R. E., Nebert D. W.,
Bigelow S. W.: Biochem. Pharmacol. 32, 3053 (1983).
WP12 THE USE OF BACTERIAL
MEMBRANES IN THE STUDY OF
INTERACTIONS OF HUMAN
CYTOCHROMES P450 AND
XENOBIOTICS
Acknowledgements:
This study was supported by grant of the Grant Agency
of Czech Republic, No.: 203/02/1152.
REFERENCES:
1. Souãek, P., Gut, I. & Stopka, P.: Chem. Biol. Interact.
126, 45 (2000).
ELI·KA KONDROVÁa,b, and PAVEL SOUâEKb
a rd
3 Faculty of Medicine, Charles University, Ruská 87,
100 00 Praha 10, Czech republic, bCenter of Industrial Hygiene and Occupational Health, National Institute of Public Health, ·robárova 48, 100 42 Praha 10, Czech Republic, [email protected]
The need to identify the participation of individual human biotransformational enzymes in the metabolism of xenobiotics has lead to the use of heterologously expressed
enzymes for these purposes. In our previous work1 we have
performed a series of experiments in which the decrease of
cytochrome P450 (CYP) content and the extent of oxidative
stress was related to the presence of metabolites of benzene
(benzoquinone, hydroquinone) and NADPH in samples containing rat and human microsomes. In order to investigate
the role of individual CYPs we decided to carry out a similar study using human enzymes expressed in bacterial membranes. Our goal therefore was to create a system suitable for
in vitro testing of interactions of CYPs and xenobiotics.
We expressed human CYPs and NADPH-CYP reductase (NPR) in Escherichia coli and isolated the membrane
fraction by differential centrifugation. The catalytic activity of the enzymes was verified and the membranes were
used directly for incubations without further purification.
After 60 minutes incubation at 37 °C in the absence or presence of NADPH and substrates (benzoquinone, hydroquinone, catechol), we measured CYP content, NPR content,
the peroxidation of lipids and the formation of hydroxyl radicals. We observed CYP destruction that was dependent
on the concentration of NADPH but as opposed to liver
microsomes we found CYP destruction in control samples
incubated without NADPH. CYP2E1 proved to be less
stable than CYP2A6 and 2C9 and therefore not suitable for
testing the effect of substrates on CYP content. We did not
detect lipid peroxidation but we were able to measure the
formation of OH radicals in the membranes. The effect of
substrates on CYP destruction, NPR destruction and oxidative stress was in agreement with the results obtained with
liver microsomes. Our results suggest that benzoquinone
and hydroquinone can initiate the generation of OH radicals and damage proteins. It is possible to conclude that human CYPs expressed in bacterial membranes are in this
form catalytically active although each CYP has different
properties and is suitable for different types of experiments. These include the measurement of oxidative stress
and with some restrictions also the measurement of the destruction of proteins.
S163
WP13 AZOLE ANTIFUNGAL DRUGS
ACT UPON CYTOCHROME P450
MONO-OXYGENASES TO
EFFICIENTLY INHIBIT
GROWTH IN MYCOBACTERIA
AND STREPTOMYCETES
KER R. MARSHALL, KIRSTY J. MCLEAN,
DAVID LEYS, and ANDREW W. MUNRO.
Dept. of Biochemistry, University of Leicester, Leicester,
UK, LE1 7RH, [email protected]
The genome sequence of several actinomycetes has revealed a surprisingly large number of genes encoding cytochromes P450 mono-oxygenases (P450s) indicative, perhaps, of essential physiological roles. Among these actinomycete P450s are homologues of 14α-sterol demethylases
Fig. 1. Chemical structures of azole antifungal drugs tested for
binding to CYP121 and potency as anti-mycobacterials.
A = clotrimazole, B = econazole, C = fluconazole, D = miconazole, E = ketoconazole.
Fig. 2. Binding of ketoconazole to Mtb CYP121 in the presence of
10 µM lanosterol. The main figure shows selected absolute
spectra induced by addition of ketoconazole to CYP121 (6.5 µM),
with progressive spectral shifts induced by addition of ketoconazole at 1.2, 2.2, 5.2 and 9.2 µM. The inset shows a plot of maximal shifts in absorption (A430 minus A408) for binding of ketoconazole to CYP121, versus the relevant [ketoconazole]. Data were fitted to a hyperbolic function. Kd = 3.3 ± 0.3 µM.
(CYP51 family) that are targets in yeasts and fungi for the
azole class of drugs (Fig. 1). Others have shown that the
Mycobacterium tuberculosis (Mtb) CYP51 homologue
binds to azole drugs 1,2. In this work we demonstrate that
a second Mtb P450 (CYP121) binds azole drugs with even
greater affinity than CYP513,4 (Fig. 2; Table 1). Growth
inhibition experiments using Mycobacterium smegmatis
(a safe laboratory model for Mtb), in the presence of a number of azole drugs, gave MIC values of <0.2 and 0.3 µM for
Tab. 1. Comparison of Kd values for CYP121 binding with MIC
values for M. smegmatis for selected azole drugs. ND = no spectral change observed. The Kd values for the binding of these drugs
to M. tuberculosis CYP51 are all in the range 5-12 µM.
econazole and clotrimazole, respectively, compared to
MIC values of 1.2 µM for rifampicin and 36.5 µM for
isoniazid, the current front-line anti-tubercular drugs of
choice3 (Table 1). Of note was the fact that Kd values for
binding of azole drugs to CYP121 mirrored MIC values in
our growth inhibition experiments3 (Table 1). Moreover,
the fact that a Streptomyces coelicolor (S. coelicolor)
CYP51 knock-out mutant remained as susceptible as wildtype to the inhibitory effects of azole drugs indicated
CYP51 is unlikely to be the primary drug target3. Similarly,
a CYP105D5 knock-out mutant of S. coelicolor we have
constructed also remains equally sensitive to growth inhibition3. An attractive scenario is that at least two Mtb
P450s are azole drug targets making the currently experienced problems with drug resistant mutants of Mtb less likely. In recent studies, we have solved the atomic structure
of Mtb CYP121 to atomic resolution - providing the basis
by which to rationalise the high affinity binding of azole
drugs5.
REFERENCES
1. Bellamine A., Mangla A. T., Nes W. D., Waterman
M.R.: Proc. Natl. Acad. Sci. USA 96, 8937 (1999).
2. Guardiola-Diaz H. M., Foster L-A., Mushrush D., Vaz
A. D. N.: Biochem. Pharmacol. 61, 1463 (2001).
3. McLean K. J., Marshall K. R., Richmond A., Hunter I.
S., Fowler K., Kieser T., Gurcha S. S., Besra G. S.,
Munro A. W.: Microbiology 148, 2837 (2002).
4. McLean K.J., Cheesman M. R., Rivers S. L., Richmond
A., Leys D., Chapman S. K., Reid G. A., Price N. C.,
Kelly S. M., Clarkson J., Smith W. E., Munro A.W.:J.
Inorg. Biochem. 91, 527 (2002).
5. Leys D., Mowat C. G., McLean K. J., Richmond A.,
Chapman S. K., Walkinshaw M. D., Munro A. W. J.
Biol. Chem. 278, 5141 (2003).
Drug
MIC for
M. smegmatis
Kd for
Mtb CYP121
Econazole
< 0.2 µM
(< 0.1 µg/ml)
< 0.2 µM
(< 0.09 µg/ml)
WP14 SUDAN I METABOLISM BY
1A1 IS STIMULATED BY
MICROSOMAL CYTOCHROME B5
Clotrimazole
0.3 µM
(0.1 µg/ml)
< 0.2 µM
(< 0.07 µg/ml)
VÁCLAV MARTÍNEK, and MARIE STIBOROVÁ
Micronazole
2.6 µM
(<1.25 µg/ml)
< 0.2 µM
(< 0.10 µg/ml)
Ketoconazole
38 µM
(20 µg/ml)
3.3±0.3 µM
(1.75±0.2 µg/ml)
>325 µM
(> 100 µg/ml)
9.7±0.2 µM
(3.0±0.1 µg/ml)
Isoniazid
36.5 µM
(5 µg/ml)
ND
Rifampicin
1.2 µM
(1 µg/ml)
ND
Fluconazole
University, Albertov 2030, 128 40 Prague 2, The Czech Republic, [email protected], [email protected]
Many cytochrome P450 (CYP) - dependent reactions
have been shown to be stimulated by another microsomal
protein, cytochrome b5. CYP3A4 - mediated reactions are
the most sensitive from this point of view. Recent work of
Guengerich and his co-workers1 has also shown stimulation of catalytic activities of CYPs 2A6, 2B6, 2C8, 2C19,
and 3A5 by cytochrome b5. On the other hand, some CYPs
S164
have not shown effects of cytochrome b5, i.e. 1A1, 1A2,
1B1 and 2D61. Here we report controversial results
showing a strong sensitivity of CYP1A1 and 1A2 to cytochrome b5.
1-Phenylazo-2-hydroxynaphthalene (Sudan I, C.I. Solvent Yellow 14) is a liver and urinary bladder carcinogen in
mammals. We found that the most efficient CYP responsible for the metabolism of this carcinogen (activation
and/or detoxication) in hepatic rat and human microsomes
is CYP1A1. In human hepatic microsomes Sudan I is also
oxidized by CYP3A4, but to a lesser extent2,3. Several
other human CYPs as 1A2, 1B1, 2A6, 2C19 and 2D6 also
oxidized Sudan I, but with efficiencies less than one order
of magnitude lower. The capabilities of some CYP enzymes to oxidize Sudan I was significantly increased by cytochrome b5. The CYP1A1, 1A2, 2A6 - dependent Sudan
I oxidation was stimulated by cytochrome b5, while reactions catalyzed by CYP1B1, 2C19, 2D6 were insensitive to
this microsomal protein. The ability of CYP3A4 to oxidize
Sudan I was almost undetectable without this protein. However, cytochrome b5 caused increase of its efficiencies to
oxidize Sudan I 3.9-fold. Stimulation of the CYP - mediated Sudan I oxidation reactions was dependent on concentration of cytochrome b5. Mechanism of cytochrome b5-dependent stimulation of the CYP catalyzed oxidation is discussed. The results presented in this study are the first
report on the stimulation of CYP1A1-mediated oxidative
reactions by cytochrome b5. Caution is highly recommended in interpretation of the effect of this microsomal protein on catalysis mediated by some CYPs in studies utilizing only one of CYP substrates.
Supported by Grant Agency of Charles University
(241/2001/B/CH/PrF) and Ministry of Education of the
Czech Republic (MSM 113100001).
REFERENCES
1. Yamazaki , H., Shimada, T., Martin, M.V., Guengerich,
F.P.: J. Biol. Chem. 276, 30885 (2001).
2. Stiborová M., Martínek V., R˘dlová H., Hodek P.,
Schmeiser H.H., Frei E.: Cancer Res. 62, 5678 (2002).
3. Martínek V., Stiborová M.: Collect. Czech. Chem.
Commun. 67, 1883 (2002).
S165
WP15 P450 AROMATASE INHIBITION
ASSAY BASED ON AN ELISA
SYSTEM
KAZUHIRO MATSUI, SHIGEAKI NISHII, TAKUYA
ISHIBASHI, and MASANORI OKA
Tsuruga Institute of Biotechnology, TOYOBO Co., LTD.,
10-24 Toyo-Cho, Tsuruga-shi, Fukui, 914-0047, Japan,
[email protected]
A convenient tool for P450 aromatase (P450arom) inhibition assay based on an Enzyme Linked Immunosorbent
Assay (ELISA) system has been established. The assay
consists of two parts. The first part is aromatase reaction,
which converts a testosterone to a 17β-estradiol, using a recombinant human P450arom and a NADPH regenerating
system. The second is an ELISA system using a highly specific and sensitive anti-estradiol monoclonal antibody immobilized on microplate and estradiol-3-CMO-horse radish peroxidase (E2-3-CMO-HRP). We could estimate
pharmaceutical aromatase inhibitors (α-naphtoflavone, ketoconazole, aminoglutethimide) and organic tins (Tributyl
tin: TBT and Triphenyltin: TPT) from their dose response
curves by this ELISA based inhibition assay. The assay
system is free from interference of fluorescence from tested chemicals themselves and does not have to use radio labeled ligands and does not have to care about radio active
wastes. It has a potential for a high throughput screening of
drug candidates and endocrine disrupting chemicals without place limitation and also without highly trained labors. Cross-reactivities of the antibody may cause some
problem in some evaluations for some chemicals. It can be
overcome by using different reagent parts (for example,
E2-6-HRP and an antibody for E2-6-BSA or anti-Estriol
antibody, Estriol-HRP and androstendione as an aromatase
substrate).
REFERENCES
1. Stresser et.al.: Anal. Biochem., 284, 427 (2000).
2. Cooke et.al.: Toxicol. Letters, 126, 123 (2002).
3. Crespi, et..al., Advances in Pharmacology, 43, 171
(1997)
4. Matsui et.al.: Abstract of Aromatase 2002 (IV International Aromatase Conference), 81 (2002)
These results, the first report on the metabolism of
2-anisidine by human enzymes, strongly suggest a carcinogenic potency of this rodent carcinogen for humans.
Supported by GACR (grant 203/03/0283) and the Ministry of Education of the Czech Republic (MSM
113100001).
WP16 CYTOCHROME P450-MEDIATED
ACTIVATION OF
CARCINOGENIC 2-ANISIDINE
LEADS TO FORMATION OF DNA
ADDUCTS IN VITRO AND
IN VIVO
WP17 POSSIBLE ROLE OF CYP450S
IN THE NABUMETONE
METABOLISM
MARKÉTA MIK·ANOVÁa, LENKA VONDRÁ·KOVÁa,
MIROSLAV ·ULCa, HEINZ H. SCHMEISERb,
EVA FREIb, and MARIE STIBOROVÁa
a
University,Albertov 2030, 128 40 Prague 2, Czech Republic, bDepartment of Molecular Toxicology, German Cancer
Reserach Center, Im Neuenheimer Feld 280, 69 120 Heidelberg, Germany
2-Anisidine is an important industrial chemical in the
manufacture of azo dyes. This chemical exhibits strong
carcinogenic activity, causing neoplastic transformation in
the urinary bladder and, to a lesser extent in spleen, liver
and kidney, of two animal species rats and mice. The mechanism of carcinogenicity of the chemical, unknown until
recent time, was studied in this work.
We found that CYP-mediated oxidation of 2-anisidine is
responsible for its metabolic activation to N-(2-methoxyphenyl)hydroxylamine as a proximate carcinogen. Using two
independent methods, namely the 32P-postlabeling technique and 14C-labeled 2-anisidine, we show for the first time
that 2-anisidine forms covalent adducts in vitro and in DNA
of target tissues (urinary bladder, and to a lesser extent, kidney, spleen and liver) in rats. The nitrenium (or carbenium)
ion is the reactive species generating covalent adducts with
deoxyguanosine in DNA in vitro and in vivo. 2-Anisidine is
also activated by peroxidases, besides CYPs.
Studies using hepatic microsomes of rats and rabbits
pre-treated with several cytochrome P450 inducers showed
that CYP2B2/4, CYP1A1/2 and CYP2E1 are mainly responsible for metabolism of 2-anisidine in these animal models. Human hepatic microsomal samples from 9 different
human donors also oxidized 2-anisidine. In order to resolve
which human CYPs are responsible for 2-anisidine oxidation several approaches were utilized: (i) correlation of
CYP-catalytic activities (or CYP contents) in each microsomal sample with the levels of a proximate carcinogenic
N-(2-methoxyphenyl)hydroxylamine metabolite formed by
the same microsomes, (ii) selective CYPs inhibition in human microsomes and (iii) microsomes of baculovirustransfected insect cells containing human recombinant
CYPs (Supersomes®). On the basis of these studies we attributed most of the 2-anisidine oxidation in human hepatic
microsomes to CYP2E1. This was confirmed with human
recombinant CYPs expressed in Supersomes, but CYP 1A2
and 2B6 are other CYP enzymes effectively oxidizing
2-anisidine.
S166
M. NOBILISa, E. ANZENBACHEROVÁb,
M. HOLâAPEKc, L. KOLÁ¤OVÁc, J. KVùTINAa,
and P. ANZENBACHERb
a
Institute of Experimental Biopharmaceutics, Joint Research Center of PRO.MED.CS Praha a.s. and Academy
of Sciences of the Czech Republic, Heyrovského 1207,
CZ-500 02 Hradec Králové, Czech Republic, bFaculty of
Medicine, Palack˘ University, Hnûvotínská 3, CZ-775 15
Olomouc,Czech Republic, cUniversity of Pardubice, Faculty of Chemical Technology, Department of Analytical
Chemistry, nám.âs. legií 565, CZ-532 10 Pardubice, Czech
Republic
The biotransformation of a non-steroidal anti-inflammatory drug (NSAID) nabumetone [4-(6-methoxy-2naphthyl)butan-2-one] in the microsomal fraction of human hepatocytes was investigated. Nabumetone is a prodrug, which is metabolized in the organism to the principal
pharmacodynamically active metabolite - 6-methoxy-2naphthylacetic acid (6-MNA). This metabolic change includes the oxidative removal of a two-carbon fragment (probably in the form of acetate) from the side chain of the nabumetone molecule. Nabumetone and its principal metabolite 6-MNA may undergo another two metabolic changes
during Phase I metabolism : a carbonyl reduction on the
side aliphatic chain of nabumetone and O-desmethylation
of nabumetone, of its reduced metabolite [4-(6-methoxy-2naphthyl)butan-2-ol] as well as of the 6-MNA. There was
no information found in the literature about the nature of
the enzymes responsible for the above-mentioned metabolic changes.
The role of CYP450s in the metabolism of nabumetone
was studied. Nabumetone and its principal metabolite,
6-MNA, were incubated with the microsomal fraction of
human hepatocytes under various conditions (coenzymes,
inhibitors, etc). Nabumetone and its metabolites in the extracts from the incubations were determined by a validated
LLE-HPLC-DAD-MS method.
The results of our experiments predicate the possible
involvement of CYP450(s) in O-desmethylation of nabumetone and its principal metabolite, 6-methoxy-2-naphthylacetic acid (6-MNA). Our results correspond with the findings that naproxen, a higher homologue of 6-MNA, un-
dergoes O-desmethylation catalysed by various isoform of
CYP4501,2.
Acknowledgment
A support from GA CR (Grant No 203/02/1152) is gratefully acknowledged.
REFERENCES
1. Miners JO, Coulter S, Tukey RH, Veronese ME, Birkett
DJ, Biochem. Pharmacol. 51, 1003, (1996).
2. Tracy TS, Marra C, Wrighton SA, Gonzalez FJ, Korzekwa KR, Eur. J. Clin. Pharmacol. 52, 293, (1997).
WP18 CYTOCHROME P450 1A2 AND
N-ACETYLTRANSFERASE
(NAT2) PHENOTYPES AS
MARKERS OF CANCER
SUSCEPTIBILITY BY WORKERS
EXPOSED TO AROMATIC
AMINES
rant cells (y = -0,0434x + 4,1419 ; r = -0,255). However, the
NAT2 activity marker correlated (y = -2,2632x + 4,1434;
r = -0,646) with percentage of abberant cells better. The
extent of percentage of abberant cells was hence shown to
be indirectly proportional to the activity of NAT2 which is
in accordance with literature. A new marker is proposed
here - ratio of CYP1A2 and NAT2 activity marker, in other
words {(17U+17X)/137X}/{AFMU/1X}. This new marker
exhibits even closer correlation (y = 0,003x + 3,2009;
r = 0,847) with found percentage of abberant cells. Although the tested group was very small, the results suggest,
that index {(17U+17X)/137X}/{AFMU/1X} could be useful for determination of cancer susceptibility in human exposed to aromatic amines as it takes into account both the
CYP1A2 and NAT2 activities.
REFERENCES
1. Butler M.A. et al.: Pharmacogenetics 2, 116 (1992)
WP19 METHOD OF PRELIMINARY
SELECTION OF SUBSTANCES
SPECIFIC IN RELATION TO
P450 ISOFORMS
JI¤Í PASTERAa, PAVEL ANZENBACHERb,
ZDENEK FIALAc, RENATA NÁDVORNÍKOVÁd,
JANA ·ALANDOVÁd, and JAROSLAV KVùTINAa
a
Institute of Experimental Biopharmaceutics, Joint Research
Center of PRO.MED.CS Praha a.s. and Academy of Sciences of the Czech Republic, Heyrovského 1207, CZ-500 02
Hradec Králové, Czech Republic, bFaculty of Medicine, Palack˘ University, Hnûvotínská 3, CZ-775 15 Olomouc, Czech
Republic, cCharles University School of Medicine, Hradec
Králové, Czech Republic, dOccupation Health Center,
Kyjevská 44, Pardubice, Czech Republic
[email protected], [email protected],
[email protected], [email protected]
The aim of this work is a proposal of the new marker for
individual susceptibility in humans exposed to aromatic
amines. A group of workers (n = 9) with professional risk
of exposition to aromatic amines and with high degree of
chromosomal damage (3 - 5 % of abberant cells) was selected. Chromosomal aberrations were evaluated using
standard method of cytogenetic analysis of peripheral
lymphocytes. Simultaneously activities of CYP1A2 and
N-acetyltransferase (NAT2) were determined using the caffeine test according to Butler et al. (1992). The CYP1A2
phenotype was expressed as the molar ratio of the sum of
1,7-dimethylxanthine [17X] and 1,7-dimethyluric acid
[17U] to caffeine [137X]. The N-acetyltransferase (NAT2)
phenotype was expressed as the molar ratio of 5-acetylamino-6-formylamino-3methyluracil [AFMU] to 1-methylxanthine [1X]. There was no correlation found between
CYP1A2 phenotypes and determined percentage of abbe-
S167
ALEXANDER A. POGREBNOYa,
VYACHESLAV E. RYABININa,
VLADIMIR A. POTEMKINb,
EKATERINA V. BARTASHEVICHb, and
MARIA A. GRISHINAb
a
Chelyabinsk state medical academy, Vorovsky Str., 64,
Chelyabinsk, 454092, Russia, bChelyabinsk State university, Br. Kashirinych Str., 129, Chelyabinsk, 454021, Russia
[email protected]
It is well known, that P450 isoforms responsible for the
metabolism of xenobiotics plays the important role in drug
interactions. Therefore, usually for drug development it
can be required that some substance would be, for example, particular isoform substrate or, on the contrary, would
not be such substrate.
The computer modeling of P450 isoforms substrate
specificity is a difficult task because of the large variety of
substrates structures. For this reason the development of
computer model capable to predict with high quality all
possible substrates of isoform can take a long time. On the
other hand, the search of substance with specified metabolic properties can be performed using more simple predicting model. A new approach for construction of such model is offered in the present paper.
A 3D-QSAR algorithm BiS1 has been used for the predicting model creating. The dataset of P450 isoforms substrates has been used as a training set. The same dataset has
been used as a testing set. The quality of the prediction for
each isoform has been estimated using two parameters. The
first parameter is quality of recognition has been defined as
a relation of number of objects correctly referred by the
prediction to total number of objects referred by the prediction to the considered class of substances. The second
parameter is dataset coverage has been defined as a relation
of number of objects correctly referred by the prediction to
total number of objects actually belonging to this class.
The classification of P450 isoforms substrates of 100%
quality of recognition has been achieved with dataset coverage about 5% for 1A2, 8% for 2A6, 40% for 2B6, 6%
for 2C, 8% for 2C19, 44% for 2C8, 22% for 2C9, 18% for
2D6, 45% for 2E1, 26% for 3A, 18% for 3A4, 33% for
3A5. The recognition of substances which are not being
P450 isoforms substrates of 100% quality of recognition
has been achieved with dataset coverage about 11% for
1A2, 31% for 2A6, 57% for 2B6, 1% for 2C, 29% for
2C19, 34% for 2C8, 15% for 2C9, 37% for 2D6, 66% for
2E1, 19% for 3A, 11% for 3A4, 35% for 3A5.
Thus, it is shown, that the substances, which have specified metabolic properties in relation to P450 isoforms,
can be revealed using the offered method. In spite of the
fact, that the offered method is able to reveal only some
part of such substances, the quality of recognition of the revealed substances in all cases achieves 100%. Hence the
offered method can be used for preliminary selection of
substances, which are specific in relation to P450 isoforms.
REFERENCES
1. Potemkin V.A., Bartashevich E.V., Grishina M.A., Guccione S.: Proc. 13th European Symp. Quantit. Structure-Activity Relationships, QSAR 2000. Heinrich-Heine-Universität, Düsseldorf, Germany, 27 August-1
September, 2000, Barcelona: Prous Science Publishers,
2001, 349-353.
S168
WP20 PEROXIDASE AND CYPMEDIATED ELLIPTICINE-DNA
ADDUCT FORMATION
EXPLAINS THE SELECTIVE
EFFICIENCY OF THIS
ANTICANCER DRUG AGAINST
BREAST CANCER AND
LEUKEMIA
JITKA POLJAKOVÁa, KRISTINA FORSTEROVÁa,
MIROSLAV ·ULCa, EVA FREIb, and
MARIE STIBOROVÁa
a
University, Albertov 2030, 128 40 Prague 2, Czech Republic, bDepartment of Molecular Toxicology, German Cancer
Research Center, Im Neuenheimer Feld 280, 61920 Heidelberg, Germany
Ellipticine and its more soluble derivatives exhibit promising results in the treatment of breast cancer and acute
myeloblastic leukemia. The inhibition of topoisomerase II
after intercalation into DNA was considered an important
property for its cytotoxicity. The CYP-dependent activation
leading to DNA adduct formation, we found to be generated
in vitro, is a novel mechanism for the ellipticine pharmacological action1,2. Target tumor cells express, beside CYP1A1,
1B1, 3A4, also peroxidases (myeloperoxidase or lactoperoxidase) in higher levels than cells of peritumoral tissues.
Since the participation of peroxidases in metabolism of
ellipticine has not been identified yet, the aim of the present
work is to extend our knowledge on this issue. Lactoperoxidase, myeloperoxidase and a model plant peroxidase (from
horseradish) were used in our study. Ellipticine is oxidized
by all these peroxidases via a radical mechanism. Using
mass spectrometry (MALDI-TOF, EI) we identify that ellipticine is primarily oxidized to a one electron reaction product (radical) producing a dimer as the major metabolite. Its
formation is inhibited by radical trapping agents (glutathione, NADH) and by DNA or deoxyguanosine 3’-monophosphate. Another ellipticine metabolite formed by peroxidases is N(2)-oxide of ellipticine. The same metabolite is
formed also by CYP-mediated reactions. During oxidation
of ellipticine by peroxidases, two ellipticine-DNA adducts,
which were generated by CYP-mediated reaction1,2, are also
formed. Identities of adducts formed by CYPs and peroxidases were confirmed by co-chromatography on HPLC. Deoxyguanosine was determined as a target deoxynucleoside for
ellipticine covalent binding in DNA. The involvement of
myeloperoxidase and lactoperoxidase in an increase of ellipticine pharmacological efficiency in the target tumor tissue
is discussed.
REFERENCES
1. Stiborová M., Bieler C.A., Wiessler M., Frei E.: Biochem. Pharmacol., 62, 1675 (2001)..
2. Frei E., Bieler C.A., Arlt V.M., Wiessler M., Stiborová
M.: Biochem. Pharmacol., 64, 289 (2002).
WP21 DISTINCT INHIBITION OF
HUMAN CYP2A6 AND MOUSE
CYP2A5 BY DERIVATIVES
OF LACTONE, PHENYLETHYLAMINE AND BENZALDEHYDE
MINNA RAHNASTOa, HANNU RAUNIOa,
ANTTI POSOb, and RISTO O. JUVONENa
Departments of aPharmacology and Toxicology, bPharmaceutical Chemistry, University of Kuopio, POB 1627,
70211 Kuopio, Finland
Aim
The purpose of this study was to test the inhibition potency of lactone, benzaldehyde and phenylethylamine derivatives against human CYP2A6 and mouse CYP2A5 and
analyse these results with Structure-Activity Relationship
(SAR) methods.
Background
The orthologous human CYP2A6 and mouse CYP2A5
enzymes metabolise a number of drugs and toxic agents.
Nicotine, the addictive component in tobacco, is mainly
metabolized to cotinine by CYP2A6. Interindividual genetic differences in CYP2A6 activity affect on nicotine kinetics, smoking behaviour and lung cancer risk. Mimicking
the CYP2A6 poor metaboliser phenotype by inhibiting
CYP2A6 enzyme in vivo results in decreased smoking and
renders nicotine orally available. Here we have determined
IC50 values of coumarin 7-hydroxylation for lactone, benzaldehyde and phenylethylamine derivatives as CYP2A5
and CYP2A6 inhibitors and analysed the results using the
Comparative Molecular Field Analysis (CoMFA) method.
Results
A rapid 96-well plate assay method was developed and
validated to measure liver microsomal coumarin 7-hydroxylation in vitro, which was used to test inhibition properties of
novel benzaldehyde and phenylethylamine derivatives. Almost all lactone derivatives inhibited more potently CYP2A5
than CYP2A6. 4-Methoxybenzaldehyde inhibited similarly
mouse CYP2A5 and human CYP2A6. All of the other benzaldehydes and phenylethylamine derivatives were more potent inhibitors of human CYP2A6 than mouse CYP2A5. The
IC50 values of 2-(p-tolyl)-ethylamine and 4-methoxybenzaldehyde were less than 10 µM for the CYP2A6 enzyme and
S169
the IC50 value of the weakest inhibitor, amphetamine, was
320 µM. Compounds with amine groups were more potent
inhibitors of CYP2A6 than CYP2A5. 4-Alkyl substitution of
benzaldehyde or 2-phenylethylamine increased the potency
of these compounds for both enzymes. Benzaldehyde was an
irreversible inhibitor of CYP2A6, since the inhibition was
dependent on both its concentration and incubation time. Presence of a methyl or methoxy substitution at the 4-position in
benzaldehyde changed the inhibition pattern, since 4-methylbenzaldehyde and 4-methoxybenzaldehyde were reversible
inhibitors. CoMFA analysis demonstrated that the active site
of CYP2A5 is larger than that of CYP2A6.
Conclusions
Inhibition potency of most tested compounds differ between the orthologous enzymes CYP2A5 and CYP2A6.
The active site of CYP2A5 is larger than that of CYP2A6,
but the size of inhibitor alone could not explain the different inhibition potencies of most lactone, benzaldehyde
and phenylethylamine derivatives. Rather, their chemical
character cause the different interactions at the active sites
of these enzymes. CoMFA analysis gave initial steric and
electrostatic field requirements for an inhibiting agent. The
most potent of these compounds could be used as lead molecules for developing potent, selective and safe CYP2A6
inhibitors for clinical use.
WP22 CHARACTERIZATION OF
HUMAN P450 INVOLVED IN
OXIDATION OF MANIDIPINE,
A 1,4 DIHYDROPYRIDINE
CALCIUM CHANNEL BLOCKER
B. RICCARDI, P. PUCCINI, D. SPINABELLI, and
D. ACERBI
Drug Metabolism and Pharmacokinetic Dept., Chiesi Farmaceutici, Via S. Leonardo 96, 43100 Parma, Italy
[email protected]
Manidipine (MAN) is a 1,4 dihydropyridine calcium
channel blocker used in the treatment of hypertension.
MAN is metabolised by oxidative metabolism; the most
important metabolic pathways are the dehydrogenation of
the 1,4-dihydropyridine ring and the oxidative cleavage of
the C-3 side chain, giving raise to MIX and MXIII metabolites. Similar metabolic pathways have been reported for
several dihydropyridines and the oxidation of dihydropyridines is known to be catalysed by CYP3A4. However, the
candidate human CYP isoform(s) responsible for MAN
metabolism has not yet been identified. In vitro studies
were then designed to clarify this point.
When MAN was incubated with 13 human liver microsomes (HLM), MAN disappearance as well as the forma-
tion of MIX and MXIII metabolites were highly correlated
with CYP3A4-linked testosterone 6β-hydroxylase activity.
Furthermore the presence of ketoconazole, a selective inhibitor of CYP3A4, effectively inhibited the disappearance
of MAN and the formation of M-IX and M-XIII in HLM.
Manidipine metabolism and M-IX and M-XIII formation
were strongly inhibited by the addition of monoclonal antiCYP3A4/5 antibodies. When MAN was incubated in the
presence of the main human recombinant CYPs, only
CYP3A4 was capable to metabolize MAN.
These results support the evidence that MAN Phase
I metabolism is mainly mediated by CYP3A4.
As the combination of a calcium channel blocker and
an angiotensin converting enzyme inhibitor is particularly
advantageous in hypertension, a fixed combination of
MAN and delapril (DEL) has been developed. Therefore, it
was evaluated if DEL could determine significant drugdrug interactions with MAN.
The in vitro addition of DEL and its active de-esterified
metabolite M-I to HLM incubations did not result in the inhibition of any of the main CYP450 linked activities. In addition, when M-I metabolism was studied, CYP3A4 did not
result to be involved in its metabolism. Therefore these
data indicate that DEL has no significant interaction with
the major CYP-450 isoforms and no metabolic interaction
is likely between manidipine and delapril.
WP23 HUMAN CYTOCHROMES P450
1A1/2 AND NADPH:CYTOCHROME P450 REDUCTASE ACTIVATE
CARCINOGENIC ARISTOLOCHIC ACID TO FORM DNA
ADDUCTS FOUND IN PATIENTS
WITH CHINESE HERBS
NEPHROPATHY
MARIE STIBOROVÁa, EVA FREIb, BRUNO SOPKOa,
KLÁRA SOPKOVÁa, VLADIMÍRA MARKOVÁa,
MARTINA LA≈KOVÁa, TEREZA KUMST¯¤OVÁa,
MANFRED WIESSLERb, and HEINZ H. SCHMEISERb
rent human livers and from one human kidney to activate
AAI, the major component of a plant extract AA, to metabolites forming DNA adducts by the 32P-postlabeling assay.
Microsomes of both these human organs generated DNA
adduct patterns reproducing those found in renal tissues
from humans exposed to AA. 7-(Deoxyadenosin-N6-yl)aristolactam I (dA-AAI), 7-(deoxyguanosin-N2-yl)aristolactam I (dG-AAI) and 7-(deoxyadenosin-N6-yl)aristolactam
II were identified as AA-DNA adducts formed from AAI
by all human hepatic microsomes. The same AA-DNA adduct pattern was observed upon incubation of AAI with
microsomes isolated from kidney of one human donor.
According to the structures of the DNA adducts identified,
nitroreduction is the crucial pathway in the metabolic activation of AAI. To define the role of human microsomal reductases in the reductive activation of AAI, we investigated the modulation of AAI-DNA adduct formation by selective inhibitors of P450s and cofactors or inhibitors of
NADPH:P450 reductase. The role of the enzymes in AAI
activation was also investigated by correlating the P450and NADPH:P450 reductase-dependent catalytic activities
in different human hepatic microsomal samples with the levels of AAI-DNA adducts formed by the same microsomal
samples. On the basis of these studies, we attribute most of
the activation of AAI in human hepatic microsomes to
P450 1A1/2, although a role of NADPH:P450 reductase
cannot be ruled out. On the contrary, NADPH:P450 reductase is more effective in AAI activation in kidney. With human recombinant enzymes, the role of both these enzymes
in AAI-DNA adduct formation was confirmed. Using difference spectroscopy and a molecular modeling and docking of AAI into the active sites of P450 1A1 and 1A2, the
binding orientation of AAI to the Fe ion of the heme structure in the active sites of both human P450s was predicted.
The results presented here demonstrate the potential of
microsomal enzymes in human liver and kidney to reductively activate carcinogenic AAI by nitroreduction.
Supported by the Ministry of Education of the Czech
Republic (grant MSM 1131 00001).
REFERENCES
1. Arlt V.M., Stiborová M., Schmeiser H.H.: Mutagenesis
17, 265 (2002).
a
University, Albertov 2030, 128 40 Prague 2, Czech Republic, [email protected], bDepartment of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been associated with the development of urothelial cancer in humans1. Understanding which
human enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual
susceptibility to this natural carcinogen. We examined the
ability of enzymes in microsome samples from eight diffe-
S170
WP24 INTERACTION OF OMEPRAZOLE AND CAFFEINE ON CYTOCHROME P450 1A2 EVALUATED
USING 13C-CAFFEINE BREATH
TEST IN RATS
ZBYNùK SVOBODAa, JAROSLAV KVùTINAa,
JAN BURE·b, and VIKTOR VO¤Í·EKb
a
Institute of Experimental Biopharmaceutics, Joint Research Centre of PRO.MED.CS Praha a.s. and the Czech
Academy of Sciences, Heyrovského 1207, Hradec Králové,
CZ-500 02, Czech Republic, bDepartment of Internal Medicine, Faculty of Medicine of Charles University, Hradec
Králové, Czech Republic, [email protected]
Omeprazole, a proton pump inhibitor, is an inducer of
human cytochrome P450 1A (CYP1A) enzymes and it
shows inhibitory effects on CYP2C19 and CYP3A41. In
this paper, a potential inhibitory effect of omeprazole on
caffeine biotransformation, a validated CYP1A2 marker,
was examined using 13C-caffeine breath test in rats. Omeprazole was administered in one group of animals at a single
oral dose of 4 mg/kg (therapeutic dose) and in the second
group at a dose of 40 mg/kg. Caffeine breath test was
performed 0.5 h after the administration of omeprazole.
13
C-caffeine was injected intravenously at a dose of
5 mg/kg. The expired air was constantly captured at selected intervals during four hours. 13CO2 was assayed by mass
spectrometry. The kinetics of expiration of 13CO2 was evaluated in comparison with the control group (without
omeprazole). Omeprazole causes slight inhibition of caffeine biotransformation in rats.
derma applanatum 8168, all strains from a collection of 17
different fungi cultures were able to deplete parathion. Three strains showing the highest activities were selected for
further studies: Bjerkandera adusta 8258, Pleurotus ostreatus 7989 and Phanerochaete chrysosporium 3641. These
strains depleted 50 to 96% of terbufos, azinphos-methyl,
phosmet and tribufos after four-days exposure to the pesticides. In order to identify the cellular localization of the
transformation activity, the extracellular and microsomal
fractions of Pleurotus ostreatus 7989 were evaluated in
vitro. While the activities of ligninolytic enzymes (lignin
peroxidase, manganese peroxidase and laccase) were detected in the extracellular fraction, no enzymatic modification of any of the five pesticides tested could be found,
suggesting the intracellular origin of the transformation
activity. In accordance with this observation the microsomal fraction was found able to transform three OPPs with
the following rates: 10 µmol mg prot-1 h-1 for phosmet,
5.7 µmol mg prot-1 h-1 for terbufos, and 2.2 µmol mg prot-1 h-1
for azinphos-methyl. The products from these reactions
and from the transformation of trichlorfon and malathion,
were identified by mass-spectrometry. These results, supported by specific inhibition experiments and the stringent
requirement for NADPH during the in vitro assays suggest
the involvement of a cytochrome P450.
WP26 DEVELOPMENT OF AN
IN VITRO REPORTER SYSTEM
TO ASSESS THE TRANSCRIPTIONAL REGULATION OF THE
HUMAN CYP1A2 GENE
FARHANA AMIN, MARGARET TOWN,
ALAN R BOOBIS, and DONALD S DAVIES
REFERENCES
1. Rost K.L. et al.: Int. J. Clin. Pharmacol. Therap. 37, 567
(1999).
WP25 MICROSOMAL TRANSFORMATION OF ORGANOPHOSPHORUS PESTICIDES BY WHITE
ROT FUNGI
RAFAEL VAZQUEZ-DUHALT, and JUAN JAUREGUI,
Instituto de Biotecnología, UNAM Apartado Postal 510-3,
Cuernavaca, Morelos. 62250 Mexico
Abstract
The enzymatic mechanism for the transformation of organophosphorus pesticides (OPPs) by different white-rot
fungi strains was studied. With the exception of Gano-
S171
Section on Clinical Pharmacology, Faculty of Medicine,
Imperial College London, Hammersmith Hospital, London
W12 0NN, UK, [email protected]
The CYP1A2 gene is well conserved amongst mammalian species. It is expressed mainly in the liver and the olfactory mucosa. CYP1A2 metabolises several heterocyclic
aromatic amines and amides and has been implicated in
their carcinogenic effects. Additionally, CYP1A2 plays
a significant role in drug metabolism. Changes in CYP1A2
expression in mice are associated with altered responses to
the toxic and carcinogenic effects of a number of chemicals
1
. Studies have also linked inter-individual differences in
CYP1A2 expression to elevated risks of colon cancer2. The
human CYP1A1 and CYP1A2 genes are present on chromosome 15 and are separated by 23 kb with no open reading frame between the two genes3. The two genes are
in opposite orientation to each other and share a common
5’-flanking region. Whilst the induction mechanism for
CYP1A1 has been relatively well established, much less is
known about the induction of CYP1A2. Analysis of the intergenic region has shown the presence of several HNF sites that may influence expression of either gene4. In addition, several potential XRE sites, a 3-MC responsive element and two AP-1 sites, thought to be specific for
CYP1A2 have been identified upstream of the transcription
start site of CYP1A2 (5, 6,7, 8). The aim of the present
study was to establish a reporter gene assay system with
a view to determining the role of these putative regulatory
elements in the expression of CYP1A2, and thus to develop a system that reflected, as closely as possible, the in
vivo transcriptional regulation of CYP1A2 . For this purpose, we have developed reporter constructs composed of
several fragments of the 5’UTR of the human CYP1A2
gene cloned upstream of a β-galactosidase reporter gene.
One such fragment, located at -2973 and -1770 bp (relative
to the transcriptional start site), contains two of the
CYP1A2 specific XRE-sites that are responsible for 3-MC
mediated enhancement of CYP1A2 promoter activity. Also
localised to this fragment are two putative AP-1 sites,
a HNF-1 site, a TATA box, C/EBP binding protein site,
a CCGG site specific for DNA methylation, and a 259-bp
positive control element with three major protein binding
elements. Another fragment located at -2973 to +901 bp incorporates binding sites for transcriptional factors such as
NF-1/CCAAT and two other HNFs upstream of the transcription start site. Also in this region is a 42bp sequence
thought to be crucial for constitutive CYP1A2 promoter
activity. It contains a TATA sequence for transcription factor IID binding, a SP-1 binding site (GC box) and
a CCAAT box. We report the results of transfection of Hep
G2 cells with these constructs and induction of the β-galactosidase activity following treatment with a panel of
known inducers such as TCDD and 3-MC. Additionally,
we shall discuss the implications for the CYP1A2 gene regulation, the potential for interaction with CYP1A1 and the
application for a test assay for novel inducers.
Acknowledgements
This work was funded by Servier, France.
REFERENCES
1. Gonzalez F.J., Kimura S.: Arch. Biochem. Biophys.
409, 153 (2003).
2. Landi M.T., Sinha R., Lang N.P., Kadlubar F.F.: IARC
Sci. Publ. 173 (1999).
3. Corchero J., Pimprale S., Kimura S., Gonzalez F.J.:
Pharmacogenetics 11, 1 (2001)
4. Gonzalez F.J. in: Cytochrome P450 (Schenkman J.B.,
Greim H., eds.), p.239. Berlin, Heidelberg: SpringerVerlag 1993.
5. Quattrochi L.C., Vu T., Tukey. R.H.: J Biol. Chem. 269,
6949 (1994).
6. Jaiswal A.K., Gonzalez F.J., Nebert D.W.: Nucleic
Acids Res. 13, 4503 (1985)
S172
7. Kubota M., Sogawa K., Kaizu Y. et. al.: J. Biochem.
110, 232 (1991)
8. Quattrochi L.C., Shih H., Pickwell G.V.: Arch. Biochem. Biophys. 350, 41 (1998).
WP27 SAR-BY-NMR WITH
CYP450ERYF: IMPLICATIONS
FOR HOMOTROPIC/HETEROTROPIC COOPERATIVITY
JED N. LAMPE, JOSH T. PEARSON,
JEFFREY S. GRINSTEAD,
MICHAEL J. DABROWSKI,
A. PATRICIA CAMPBELL, and WILLIAM M. ATKINS
Department of Medicinal Chemistry, Box 357610, University of Washington, Seattle, WA 98195, [email protected]
Allosteric phenomena have long been recognized in
P450 isozymes, but the molecular mechanisms are not well
understood. Recently, CYP450eryF, a prokaryotic P450
isolated from the soil bacterium Saccharopolyspora erythraea, has been shown to exhibit allosteric binding with
the ligands androstenedione, 9-aminophenanthrene, and
1-pyrenebutanol. Although some ligand-bound crystal
structures are available, they are not sufficient to describe
all the observed allosteric phenomena. In an attempt to better understand P450 allosteric dynamics, we plan to use
2D 1H-15N-HSQC-NMR to monitor the ligand-dependent
chemical shifts and peak intensities of both the bound and
unbound enzyme. To this end, CYP450eryF was expressed
in DL-39, a tyrB- ilvE- aspC- auxotrophic strain of E. coli,
using minimal media supplemented with 15N-Phe. The purified protein was confirmed have uniform isotopic labeling with 15N-Phe using LC-ESI mass spectrometry. In addition, CO difference spectra showed a characteristic peak
at 450 nm, indicating presence of the holoenzyme. 1H-15NHSQC spectra were obtained from an 800 µM sample of
15
N-Phe CYP450eryF in the presence and absence of testosterone using a 500 MHz Varian Inova NMR spectrometer. Chemical shift perturbation mapping of the 15N-edited
HSQC spectra will allow for: (1) a determination of which
of the three active site Phe residues (F86, F78, and F187)
that are critical for substrate binding, and (2) an assessment
of whether the pattern of perturbed Phe residues changes
upon addition of a second ligand, thus providing a structural probe for the observed allosteric behavior of
CYP450eryF.
WP28 IMPROVING THE AFFINITY OF
ADRENODOXIN FOR ITS
PARTNER PROTEINS BY
DIRECTED EVOLUTION
WP29 NOVEL REVERSIBLE
ADDUCTION OF THE P450
HEME: INACTIVATION OF
CYTOCHROME P450 2E1
T303A BY TERT-BUTYL
ACETYLENE
ANDREAS BICHET, FRANK HANNEMANN,
and RITA BERNHARDT
Saarland University, FR 8.8 Biochemistry, Postfach 151150,
66041 Saarbrücken, Germany, [email protected]
While over the last twenty years protein engineering
based on site-directed mutagenesis has contributed to our
understanding of enzyme catalysis, biological design capabilities have been greatly enhanced recently by the development of ‘evolutionary’ protein design methods which
use random mutagenesis followed by screening or selection for a desired trait1,2.
We applied this technique for adrenodoxin (Adx),
a [2Fe - 2S] ferredoxin involved in steroid hormone biosynthesis in the adrenal gland of mammals. It is a small soluble protein that transfers electrons from adrenodoxin reductase (AR) to different cytochrome P450 isoforms where
they are consumed in hydroxylation reactions3.
The screening-system chosen for the directed evolution
of Adx is the yeast two-hybrid system, a technique detecting interactions between two proteins4. It was developed
to provide a genetic mean of identifying proteins that physically interact in vivo. Here we describe its application as
screening-system in directed evolution, which we adapted
to our purposes to screen new adrenodoxin forms with improved affinity to its redox partners. With this system we
screened a library of 5000 Adx variants and selected new
forms. The characteristics of these forms with regard to affinity and activity comparing to Adx wild type are presented in this poster.
REFERENCES
1. Arnold, F.H. Nature 409, 253 (2001)
2. Koeller, K.M. and Wong, C.H. Nature 409, 232(2001)
3. Grinberg, A.V.; Hannemann, F.; Schiffler, B.; Müller,
J.J.; Heinemann, U. and Bernhardt, R. Proteins 40, 590
(2000)
4. Fields, S. and Song, O. Nature 340, 245 (1989)
S173
ANNA L. BLOBAUM, UTE M. KENT, and
PAUL F. HOLLENBERG.
Department of Pharmacology, University of Michigan,
1150 West Medical Center Drive, Ann Arbor, Michigan,
48109, [email protected].
The constitutively expressed and ethanol-inducible cytochrome P450 2E1 catalyzes the oxidation of a large number of drugs and hepatotoxic xenobiotics, including halocarbon anesthetics and acetaminophen, and carcinogens,
such as dialkylnitrosamines. The kinetics for the inactivation of cytochrome P450 2E1 by tert-butyl acetylene (tBA)
and tert-butyl 1-methyl-2-propynyl ether (tBMP) were previously characterized1. The inactivation of P450 2E1 by
tBA was shown to be a result of a combination of heme alkylation and protein adduction, while inactivation by the
structurally similar tBMP was a result of only heme alkylation1.
Mutation of a highly conserved threonine (T303) in the
I helix of P450 2E1 has suggested roles for this residue in
substrate interactions and orientation within the enzyme
active site and as a possible proton donor in acid-base reactions. In order to investigate the role of T303 in the inactivation of P450 2E1 by acetylenic compounds, the kinetics for the inactivation of cytochrome P450 2E1 T303A by
tBA and tBMP were characterized. The inactivation was
NADPH-dependent and proceeded in a time- and concentration-dependent manner. The losses in P450 2E1
T303A enzyme activity occurred with concurrent losses in
the P450 CO spectrum and P450 heme. These losses were
accompanied by the appearance of two different tBA- or
tBMP-modified heme products as detected by HPLC analysis. Electrospray ionization liquid chromatography mass spectrometry (ESI-LC-MS) of the modified hemes
showed masses of 661 Da or 705 Da, consistent with the
mass of an iron-depleted heme plus the masses of either the
tBA or tBMP reactive intermediate and one oxygen atom,
respectively. Surprisingly, the activity and CO spectral losses observed with the tBA-inactivated T303A mutant were
reversible with extensive dialysis. Similarly, HPLC analysis of the tBA-inactivated T303A mutant showed a decrease in the alkylated heme products and a significant recovery of the native heme after dialysis. The pH-dependence
of the reversibility was investigated by dialyzing the inactivated samples against low and neutral pH buffers. Reduced levels in the alkylated products were again observed
with concurrent increases in the native heme peak, suggesting that pH does not affect the reversibility.
The comparative studies on the inactivation of P450s 2E1
and 2E1 T303A by tBA and tBMP suggest the existence of
three distinct mechanisms for inactivation: 1) covalent alkylation of the heme prosthetic moiety, 2) a combination of
heme alkylation and protein adduction, and 3) a novel reversible alkylation of the P450 heme. These data support the notion that certain aspects of the structure of the inactivator and
the identities of critical amino acid residues within the enzyme active site may significantly influence metabolism and
therefore the inactivation of P450s 2E1 and 2E1 T303A by
tert-butyl acetylenes.
Supported in part by NIH Training Grant GMO7767.
REFERENCES
1. Blobaum, A. L., Kent, U. M., Alworth, W. L., and Hollenberg, P. F.: Chem. Res. Tox. 15, 1561 (2002).
ture and the yield of purified enzyme was nearly 75% of
the starting amount. The enzyme metabolised erythromycin forming 7.1 ± 0.9 nmol of formaldehyde/ min/ nmol of
P450. These rates are comparable to those obtained with
a human CYP3A4/ human NADPH-P450 reductase fusion
protein constructed by Shet and co-workers1. Oxygen consumption rates were 13.5 nmol of oxygen/ min/ nmol P450
in the presence of substrate and 3.2 nmol of oxygen/ min/
nmol of P450 in the absence of substrate. Further characterisation of this and other human P450/ CYP102 reductase
is currently underway.
These artificial fusion proteins provide a means to facilitate studies of these enzymes in solution as well as crystallisation screening leading to human P450 structures
where the catalytic domain has not been altered by mutagenesis.
REFERENCES
1. Shet M. S., Fisher C. W., Holmans P. L., Estabrook
R. W.: PNAS. 90, 11748 (1993).
WP30 PROTEIN ENGINEERING OF
HUMAN CYTOCHROME
P450S FOR STRUCTURE
DETERMINATION
WP31 STUDIES ON INTERACTION
BETWEEN FIBRATES AND
STATINS
VIKASH DODHIAa, RICHARD BAZINb,
and GIANFRANCO GILARDIa
a
Imperial College London, Exhibition Road, London, United Kingdom, b Molecular Informatics, Structure & Design,
Pfizer Global R&D, Kent, United Kingdom
[email protected]
The 53 human P450s that have been identified play
a critical by catalysing reactions involved in xenobiotic
metabolism, biosynthesis of steroid hormones, oxidation of
unsaturated fatty acids and metabolism of fat-soluble vitamins. However these enzymes are membrane bound and
once detached from the membrane require the presence of
detergent to keep them in an active state making studies
with these enzymes problematic. These enzymes are not
catalytically self-sufficient and as such necessitate accepting electrons from their membrane bound redox partner
NADPH-cytochrome P450 reductase.
Here we report the rational design of human cytochrome
P450s into soluble, catalytically self-sufficient chimeras
using the reductase bacterial cytochrome P450 CYP102. Fusion of N-terminally modified human P450s to the large and
negatively charge reductase domain of CYP102 (P450BM-3)
led to active enzymes (P450/BMR) that still retained a small
degree of membrane association. Nevertheless upon removal
from bacterial membranes, the enzymes remained stable in
detergent free buffer and could be purified easily using
a single anion-exchange column. This approach resulted in
the soluble chimeras CYP2C9/BMR, CYP2C19/BMR,
CYP2D6/BMR and CYP3A4/BMR. The CYP3A4/BMR
was expressed to a yield of ≈ 830 nmol/ L of bacterial cul-
S174
HIDEKI FUJINO, SYUNSUKE SHIMADA,
IWAO YAMADA, MASARU HIRANO,
YOSHIHIKO TSUNENARI, and JUNJI KOJIMA
Tokyo New Drug Research Laboratories I, Kowa Company
Ltd.,2-17-43 Noguchicho, Higashimurayama, Tokyo, Japan,
[email protected]
Recently, cerivastatin was withdrawn from the market
due to disproportionate numbers of fatal rhabdomyolysis
among patients who had received gemfibrozil concomitantly1). To gain a better understanding of the mechanism
of drug-drug interaction between fibrates and statins, several experiments in vitro were performed. A remarkable metabolic inhibition of cerivastatin and atorvastatin was noted
in the presence of gemfibrozil. However, the increase in
unchanged form was fairly small for pitavastatin, compared with other statins. An extensive metabolism of gemfibrozil was noted in human hepatic microsomes. CYP1A2,
CYP2C9 and CYP2C19 were shown to be principally responsible for the metabolism of gemfibrozil. However, no
metabolism via CYP was observed on fenofibrate, bezafibrate, cipfofibrate and clofibrate. Gemfibrozil inhibited several CYP isoforms in contrast to other fibrates. In the Dixon plots, gemfibrozil was shown to be a potent competitive inhibitor of CYP2C9-mediated metabolism with an
apparent Ki value of 18.6 µM. In contrast, gemfibrozil inhibited the CYP2C8- and CYP3A4-mediated metabolism
non-competitively with apparent Ki values of 55 and
171 µM, respectively. Although, the mechanism of the
drug-drug interaction was not completely clarified, we propose that the increase in creatine phosphokinase caused by
co-administration of gemfibrozil and statins is at least partially due to a CYP-mediated inhibition.
REFERENCE
1. Lau T.K., Leachman D.R.,: Tex Heart Inst J, 28, 142
(2001).
WP32 THE EFFECTS OF CHRONIC
TREATMENT WITH
ANTIDEPRESSANT DRUGS ON
THE LEVEL AND ACTIVITY OF
CYP2C6, CYP2C11 AND
CYP3A1/2 IN THE RAT LIVER
SSRIs. The activities of CYP2C6 and CYP2C11 were elevated by imipramine, desipramine and SSRIs. Moreover,
chronic mirtazapine increased the activity of CYP2C6,
while nefazodone did not exert any effect on the three studied isoforms. At the same time, the investigated antidepressants (TADs, SSRIs, mirtazapine) increased the hepatic level of the cytochrome proteins. Thus, the elevated level of CYP3A1/2 protein by TADs corresponded negatively with a reduced CYP3A1/2 activity by these antidepressants, while in other cases positive correlations between the level and activity of CYP isoforms were observed, i.e. an increased level of cytochrome protein was
accompanied by enhanced enzyme activity. In conclusion,
the obtained results indicate induction of CYP2C6,
CYP2C11 and CYP3A1/2 by many investigated antidepressants. However, an induction of CYP3A1/2 (an increase in protein level) by TADs seems to be masked by some
other mechanisms (e.g. formation of CYP3A-TAD reactive
metabolite complexes), which leads to decreased enzyme
activity.
ANNA HADUCH, and W. ANNA DANIEL
Department of Pharmacokinetics and Drug Metabolism,
Institute of Pharmacology, Polish Academy of Sciences,
Sm´tna 12, 31-343 Kraków, Poland
[email protected]
Antidepressants are given to patients for months or years, which creates possibility of profound alterations in hepatic enzyme system. Cytochrome P-450 subfamilies
CYP2C and CYP3A play an important role in the biotransformation of many drugs and other xenobiotics. They also
mediate the metabolism of endogenous substrates, such as
steroids. Those subfamilies constitute the substantial pool
of cytochrome P-450 in the human and rat livers.
The aim of present study was to investigate the influence of chronic treatment with tricyclic antidepressant
drugs (TADs), selective serotonine reuptake inhibitors (SSRIs) and novel drugs (mirtazapine, nefazodone) on the hepatic level and activity of CYP2C6, CYP2C11 and
CYP3A1/2 in the rat. The effect of antidepressants was studied in vitro in liver microsomes of control rats and of animals treated chronically (for two weeks, twice a day) with
pharmacological doses of the drugs (imipramine, amitriptyline, clomipramine, nefazodone - 10 mg/kg ip.; desipramine, fluoxetine, sertraline - 5 mg/kg ip.; mirtazapine 3 mg/kg ip.). Cytochrome P-450 activity was assessed by
measuring the rate of testosterone hydroxylation in positions 2α and 16α (CYP2C11), 2β and 6β (CYP3A1/2), and
the rate of warfarin 7-hydroxylation (CYP2C6). The
amount of the metabolites formed in vitro was assayed
using the HPLC method. The hepatic level of cytochrome
P-450 proteins was determined by Western blotting using
specific polyclonal antibodies.
The activity of CYP3A1/2 was significantly decreased
by chronic treatment with TADs, while it was enhanced by
S175
WP33 THE OPTICAL BIOSENSOR
STUDY OF REDOX PARTNERS’
INTERACTIONS IN THE P4502B4
SYSTEM IN HYDROXYLATION
CONDITIONS
IVANOV YU.D., KUZNETSOV V.YU., PETUSHKOVA
N.A., KANAEVA I.P,. and ARCHAKOV A.I
V.N.Orekhovich Institute of biomedical chemistry RAMS,
Pogodinskaya str., 10, Moskow, Russia
[email protected]
Investigated were the interactions between cytochrome
P4502B4 (d-2B4), NADPH cytochrome P450 reductase
(d-Fp) and cytochrome b5 (d-b5) within a monomeric reconstituted P4502B4-containing monooxygenase system in
hydroxylation conditions and in the absence of phospholipid. It was shown that immobilization of the partner
through their respective amino groups onto the carbodextran surface of the IAsys optical biosensor was accompanied by occurrence of the 7-pentoxyresorufin O-dealkylation reaction in the monomeric reconstituted (in 500 mM
K-phosphate buffer) P4502B4 system. Under such conditions, no complex formation between the reductase and
cytochrome b5 was observable. At the same time the
binary d-Fp/d-2B4 complexes were formed with the complex association/dissociation rate constants (kon and koff) =
(3±2)*104 M-1s-1 and 0.05±0.03 s-1 , respectively. The formation of ternary d-Fp/d-2B4/d-b5 complexes with the
lifetime of about 40 s was also registered.
This work was supported by RFBR grant N 01-0448245, Intas N 01-470
WP34 ATOMIC FORCE
MICROSCOPY(AFM) AND
OPTICAL BIOSENSOR STUDIES
OF CYTOCHROME P450’S
COMPONENT INTERACTIONS
WP35 METABOLISM OF N-BENZYL-1AMINOBENZOTRIAZOLE BY
P450 2B1 RESULTS IN A
MODIFIED ENZYME CAPABLE
OF N-DEMETHYLATION BUT
WITH ABOLISHED
HYDROXYLATION ACTIVITY
YURI D. IVANOVa, IRINA P. KANAEVAa,
IRINA I. KARUZINAa, KUZNETSOV V.YU.a,
SERGEY A. USANOVb, GASTON HUI BON HOAc,
STEPHEN G. SLIGARd, and
ALEXANDER I. ARCHAKOVa
UTE M. KENTa, REBECCA A. ROOFa, LISE
PASCUALa, DAVID P. BALLOUb, and
PAUL F. HOLLENBERGa
a
a
V.N.Orekhovich Institute of biomedical chemistry RAMS,
Pogodinskaya str., 10, Moscow, Russia, bInstitute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk, Belarus, cInstitut de Biologie Physico Chimique, Paris, 75005 Paris, France, dUniversity of Illinois,
Urbana, 61801, USA, [email protected]
It is shown that BIA-RM enables to reveal the binary as
well as ternary complexes between redox proteins of
P450cam, P402B4 and P450scc systems and measure kinetic
parameters of protein-protein interaction. The AFM technique allows visualize these complexes. The AFM images of
cytochrome P450cam (P450cam), putidaredoxin (Pd) and putidaredoxin reductase (PdR), binary (Pd/PdR, P450cam/Pd,
P450cam/PdR) and ternary (PdR/Pd/P450cam) complexes
were obtained and the heights on a mica support were measured. The AFM images of the redox proteins of P4502B4
system - cytochrome P4502B4 (d-2B4), NADPH-dependent cytochrome P450 reductase (d-Fp) and cytochrome b 5
(d-b5) and images of the binary complexes d-Fp/d-2B4 and
d-2B4/d-b5 were obtained on HOPG. However, we failed to
detect formation of d-Fp/d-b5 complex. The ternary d-Fp/d2B4/d-b5 complexes were visualized and the typical height
of the complexes were found to be 6.2 nm. The AFM images of the redox proteins of P450scc system were obtained
and the heights of the binary complexes AdR/P450scc,
AdR/Ad and Ad/P450scc were found to be 3.2-3.4, 3.1-3.4
and 2.8-3.6 nm, respectively.
This work was supported by RFBR grant N 01-0448245, Intas N 01-470
Department of Pharmacology and bDepartment of Biochemistry, University of Michigan, Ann Arbor, MI 48109, USA,
[email protected]
The 7-ethoxy-4-(trifluoromethyl)coumarin (EFC) activity of P450 2B1 is inactivated by N-benzyl-1-aminobenzotriazole (BBT) in a mechanism-based manner. Approximately 25% of the loss in enzymatic activity could be
accounted for by a minor decrease in the P450 CO spectrum and the formation of a small amount of an MI complex. The majority of the inactivation was due to the formation of a covalent BBT adduct. Concurrently, the mass
of the P450 apo-protein increased by 225 Da. Mechanistic
studies that examined the rates of reduction, oxy-ferro intermediate formation, and product formation suggested
that the reductive process was affected. However, the reduced rate of reduction alone could not account for the overall loss in EFC enzymatic activity. The BBT-inactivated
enzyme also exhibited a comparable loss in testosterone
hydroxylation activity. However, the ability of the BBT-inactivated enzyme to generate formaldehyde from benzphetamine metabolism was minimally affected. Substrate binding studies with benzphetamine and testosterone indicated
that the BBT-modified P450 2B1 displayed similar spectral
binding properties when compared to the native enzyme.
Analysis of the benzphetamine metabolites showed a greater propensity of the BBT-modified enzyme towards the
N-demethylation pathway and away from generating hydroxylated products. In addition, a change in the ratio of
amphetamine to methamphetamine products was observed.
With EFC or testosterone as a substrate the major defect
appears to be a reduced rate of electron transfer and an inability of the enzyme to form hydroxylated products. In
contrast to EFC and testosterone, benzphetamine can be
metabolized by the BBT-modified enzyme via N-demethylation and N-debenzylation to levels comparable to the unmodified, control enzyme. (Supported by CA 16954 from
the National Cancer Institute).
REFERENCE
Kent U.M., Bend J.R., Chamberlin B.A., Gage D.A.,
and Hollenberg P.F.: Chem. Res. Toxicol. 10, 600 (1997).
S176
WP36 A PROTON-SHUTTLE
MECHANISM MEDIATED BY
THE PORPHYRIN IN BENZENE
HYDROXYLATION BY
CYTOCHROME P450 ENZYMES
WP37 EFFECT OF MEMANTINE ON
IN VITRO CYTOCHROME
P450-MEDIATED METABOLIC
ACTIVITIES IN HUMAN LIVER
MICROSOMES
SAM P. de VISSER, and SASON SHAIK
S. MICUDAa, L. MUNDLOVAa,
E. ANZENBACHEROVAb, P. ANZENBACHERb,
J. CHLADEKa, and J. MARTINKOVAa
The Hebrew University of Jerusalem, 91904 Jerusalem,
Israel.
One of the typical reactions catalyzed by Cytochrome
P450 is arene hydroxylation. Although, the mechanisms
have been studied thoroughly by experimental means,1,2 it
is still under debate. One of the issues of controversy is the
observation of side-products such as arene oxide and cyclohexenone, of which the latter one is proposed to be obtained as the result of the so-called NIH-shift.3 This has
prompted us to carry out a quantum chemical study into the
mechanism of benzene hydroxylation by a Cpd I model.4
The calculations show a competition between radicalar and
electrophilic mechanisms in which the π-system is attacked
by the oxygen atom of the iron-oxo moiety to form two
type of σ-complexes, one radicalar and one cationic. The
dominant channel is the electrophilic substitution channel
that produces a cationic σ-complex. Both type of σ-complexes can give arene oxide products via ring-closure. This
product does not rearrange to form either phenol or cyclohexenone, so is an enzymatically dead-end product. We
discovered a mechanism for an enzymatic mechanism to
produce phenol or cyclohexenone via a proton-shuttle mechanism from the intermediate complexes.
REFERENCES
1. Korzekwa, K. R.; Swinney, D. C.; Trager, W. T. Biochemistry 28, 9019 (1989).
2. Rietjens, I. M. C. M.; Soffers, A. E. M. F.; Veeger, C.;
Vervoort, J. Biochemistry 32, 4801 (1993).
3. Jerina, D. M.; Daly, J. W. Science 185, 573 (1974).
4. de Visser, S. P.; Shaik, S. J. Am. Chem. Soc., submitted.
S177
a
Department of Pharmacology, Charles University in Prague, Faculty of Medicine in Hradec Kralove, Simkova 870,
PO Box 38, 500 38 Hradec Kralove, Czech Republic,
b
Faculty of Medicine, Palack˘ University, Hnûvotínská 3,
CZ-775 15 Olomouc, Czech Republic,
Memantine is an uncompetitive N-methyl-D-aspartate
(NMDA) receptor antagonist with therapeutic potential in
dementia, spasticity and Parkinson’s disease. This compound is predominantly excreted unchanged via the kidneys.
Nevertheless, 10% to 25% of administered dose is excreted
into bile, with 1% being metabolized to hydroxy metabolite.
Therefore, potential for drug-drug interactions at the level of
hepatic metabolism exists. This study was undertaken to
examine the effects of memantine on several drug metabolizing CYP enzymes found in the liver. Pooled human liver
microsomes were co-incubated with memantine and probe
substrates for CYP1A2 (ethoxyresorufin), CYP2A6 (coumarin), CYP2C9 (tolbutamide), CYP2D6 (dextromethorphan),
CYP2E1 (chlorzoxazone), and CYP3A4 (nifedipine). The
formation of CYP-specific metabolites following co-incubation with various memantine concentrations was determined
to establish IC50 and Ki values for these enzymes. While memantine did not inhibit CYP1A2, CYP2A6, CYP2C9,
CYP2E1, and CYP3A4 activities at concentrations below
500 microM, this compound inhibited CYP2D6 with IC50
and Ki values of 368.7 µM and 250 µM, respectively. In
conclusion, these results indicate that, although memantine
can inhibit CYP2D6 catalytic activity, it would not be expected to cause any significant interactions with others
CYP-metabolized drugs at clinically relevant concentrations
achieved during long-term therapy.
This study was performed within the framework of the
COST B15.30 project.
WP38 21-FLUOROSTEROIDS USED IN
KINETIC AND 19F-NMR
STUDIES TO PROBE CYP17
CATALYSIS
WP39 EFFECTS OF THALIDOMIDE
ON THE CYP-MEDIATED
METABOLISM OF CYCLOPHOSPHAMIDE IN THE MOUSE
EMILY O’DONNELLa, J. NEVILLE WRIGHTb,
and PETER LEE-ROBICHAUDa
JAMES PAXTONa, PHILIP KESTELLb, QI DINGa,b,
LAI-MING CHINGb, BRIAN D. PALMERb,
SHUFENG ZHOUa, and BRUCE C. BAGULEYb.
a
Centre for Chemical Biology, Dept. of Chemistry, University of Sheffield, Sheffield, S3 7HF, U.K.; bDept. of Biochemistry, University of Southampton, Southampton, SO16
7PX, U.K., [email protected]
Porcine microsomal cytochrome P450 17α-hydroxylase-17,20-lyase (CYP17) catalyses the conversion of
pregnenolone (preg) to 17α-hydroxypregnenolone which is
then a substrate for the enzyme’s cleavage activity, resulting in the production of dehydroepiandrosterone with the
side-chain being removed as acetic acid. In the presence
of cytochrome b5 (b5) preg can be converted directly to
3β-hydroxyandrosta-5,16-diene (5,16-diene)1. We have
shown that the substrate analogue, 3β-hydroxyandrost-5ene-17β-carbaldehyde (aldehyde) is cleaved directly, in the
absence of b5, to the 5,16-diene at a rate 8-fold higher than
preg2. This was attributed to enhanced reactivity of the aldehyde carbonyl with a nucleophilic heam-iron-peroxy
species. We now report that 21-fluoropreg is a substrate for
CYP17 with the side-chain being removed as fluoroacetate.
In contrast to the aldehyde, the 21-fluorocompound is cleaved at one third the rate of preg and the cleavage is not directed exclusively to the 5,16-diene. The reactivity of the
aldehyde is, therefore, unlikely to be solely due to the enhanced electrophilicity of the C-20 carbonyl but may also
be a result of an altered binding conformation. The lack of
the 21-methyl group may facilitate the juxtaposition of the
aldehyde carbonyl for attack by the active site nucleophilic
iron-peroxy intermediate, which leads to the cleavage of
the formyl side chain.
Also presented are preliminary Fluorine-NMR experiments that show that it is possible to measure the paramagnetic effect of the active site, high-spin iron on the T2 relaxation rates of the fluoropreg enzyme-bound substrate. The
data will be used to determine the distance from the activesite iron to the fluorine label in the substrate. The study can
be extended with the use of substrates, labelled with fluorine at different positions, to determine the orientation of
substrate binding.
REFERENCES
1 Nakjin S and Hall P.F.: J. Biol. Chem. 256, 3871 (1981).
2 Lee-Robichaud P., Shyadehi A.Z., Wright J.N., Akhtar
M.E., and Akhtar M.: Biochemistry 34, 14104 (1995).
S178
a
Department of Pharmacology and Clinical Pharmacology,
Auckland Cancer Society Research Centre, The University
of Auckland, Auckland, New Zealand
[email protected]
b
Concurrent administration of racemic thalidomide (TH)
(20 mg/kg, i.p.) potentiated the antitumour effect of cyclophosphamide (CP) (220 mg/kg, i.p.) with no increase in
toxicity in the murine Colon 38 tumour model. This was
accompanied by an increase in the area under the plasma
concentration-time curve (AUC) for CP (3.5-fold), 4-hydroxycyclophosphamide (4-OH-CP) (2.7-fold), dechloroethylcyclophosphamide (dCE-CP) (1.8-fold) and 4-ketocyclophosphamide (4-keto-CP) (1.3-fold), but with no significant change in their Cmax1. The aim of this study was to investigate whether the pharmacokinetic interaction arose
from TH inhibition of the major metabolic pathways of CP
associated with CYP-mediated 4-hydroxylation or dechloro-ethylation. In addition, the possibility that a TH metabolite or hydrolysis product was responsible for this interaction was investigated, as both metabolism and hydrolysis pathways are of major importance in the elimination
of TH in the mouse2.
Methods
CP, 4-OH-CP and dCE-CP were measured using solid
phase extraction and LC-MS methods1. In the in vivo studies, C57B1/6 male mice (30 - 35 g) received i.p. injections of CP (220 mg/kg) followed by TH (20 mg/kg), with
3 mice used for each time point. Having established the
concentration-time profile for 4-OH-CP with and without
TH, a 60 min blood collection time point was selected to
study the effects of 20 mg/kg of 5-hydroxy-thalidomide
(5-OH-TH) (the major CYP-mediated metabolite), α-aminoglutaramide (AG), and phthaloylisoglutamine (PG) (the
major TH hydrolysis product). At 60 min, plasma was collected for 4-OH-CP measurement. In vitro studies of CP
oxidation were undertaken with mouse liver microsomes
(0.5 mg/ml protein, 5 mM MgCl2, 1 mM NADPH, CP
[20 - 2000 µM] in pH 7.4 phosphate buffer) incubated
for 30 min at 37o. This was followed by the addition of
500 µL methanol and 200 µL semi-carbazide, and the formation of 4-OH-CP measured. The effects of TH, 5-OHTH, and PG (all at 50 and 500 µM) on the formation rate of
4-OH-CP were investigated. The formation of dCE-CP was
also investigated in a separate set of incubations in a similar manner, except that the semi-carbazide was omitted.
A reduction of > 30% in the metabolite formation rate was
considered significant.
Results
All compounds, except PG, caused a >2-fold increase
in the plasma concentrations of 4-OH-CP at 60 min. Using
1 mM CP as substrate, the rate of formation of 4-OH-CP
was not influenced by co-incubation with TH, 5-OH-TH or
PG. In contrast, there was a significant reduction in the formation rate of dCE-CP in the presence of TH. Our results
suggest that 5-OH-TH and hydrolysis products may play
a part in the pharmacokinetic interaction with CP, but that
the destruction of the glutarimide ring eliminates this activity. Direct inhibition of CYP-mediated CP activation
pathway to 4-OH-CP does not appear to play a part in this
interaction, but the deactivation pathway to dCE-CP may
be involved.
oxygen species and in the interaction with some xenobiotic
compounds such as quinones1-3.
Nilutamide 1 is a nonsteroidal antiandrogen derivative
behaving as a competitive antagonist of the androgen receptors4. This nitroaromatic compound is proposed in the
treatment of metastatic prostatic carcinoma. After oral administration in humans, Nilutamide is extensively metabolized in the liver, undergoing mainly reduction of its nitro
group to primary amine, 25. Therapeutic effects of nilutamide are overshadowed by the occurrence of several adverse reactions. More serious complications include druginduced hepatitis and pulmonary interstitial fibrosis. Absence of hypersensitivity manifestations suggests that Nilutamide hepatitis is mediated by a toxic mechanism which
remains poorly known6. Previous studies have shown that
NOSs could be involved in these toxic effects of Nilutamide7.
REFERENCES
1. Ding Q., Kestell P., Baguley B.C., Palmer B., Paxton
J.W., Muller G., Ching L-M: Cancer Chemother Pharmacol 50, 186 (2002).
2. Lu J., Palmer B.D., Kestell P., Browett, P., Baguley B.C.,
Ching L-M.: Clinical Cancer Research (in press).
Fig. 1.
WP40 REDUCTION OF THE NITROAROMATIC ANTIANDROGEN
NILUTAMIDE BY NO SYNTHASES. IMPLICATIONS FOR THE
ADVERSE EFFECTS OF THIS
ANTIANDROGEN DRUG
SYLVIE DIJOLSa, KJETIL ASKb, CLAIRE GIROUDa,
LUDIVINE CASSEa, YVES FRAPARTa,
MARIE-AGNES SARIa, PHILIPPE CAMUSb, DENNIS
STUEHRc, DANIEL MANSUYa, and
JEAN-LUC BOUCHERa.
a
Laboratoire de Chimie et Biochimie Pharmacologiques et
Toxicologiques, Université R. Descartes, UMR 8601
CNRS, 45 rue des Saints Pères, 75270 Paris cedex 06,
France, [email protected]. bLaboratoire de Pharmacologie et Toxicologie Pulmonaires, Faculté
de Medecine et de Pharmacie, 7 Boulevard Jeanne D’Arc,
21000 Dijon cedex, France. cDepartment of Immunology,
Lerner Research Fundation, Cleveland Clinic, 9500 Euclid
Avenue, Cleveland, OH 44195, USA.
Nitric oxide synthases (NOSs) are flavohemeproteins
that catalyze the oxidation of L-arginine to L-citrulline
with formation of the widespread signal molecule NO. Beside their intrinsic biological role in NO synthesis, these
enzymes are also involved in the formation of reactive
S179
We studied the ability for recombinant NOSs to interact
with Nilutamide and we showed that NOSs reduce this nitroaromatic compound with selective formation of the corresponding hydroxylamine 3. This new reaction of neuronal NOS (nNOS) proceeds with intermediate formation of
a nitro anion free-radical easily observed by EPR. The reduction of Nilutamide catalyzed by nNOS was insensitive
to the addition of usual heme ligands and L-arginine analogues but strongly inhibited by O2 and a flavin/NADPH
binding inhibitor. Involvement of the reductase domain of
the nNOS was further confirmed by the stimulating effect
of Ca2+/Calmodulin on the accumulation of hydroxylamine
and nitro anion radical and by the ability of the isolated reductase domain of nNOS to catalyze this reaction. Inducible and endothelial NOS also displayed this nitroreductase activity, albeit with lower yields.
Selective reduction of Nilutamide to an hydroxylamine
by NOSs could explain some of the toxic effects observed
with this nonsteroidal antiandrogen and other nitrocompounds.
REFERENCES
1. Alderton W.K., Cooper C.E., Knowles R.G.: Biochem.
J. 357, 593 (2001).
2. Kumagai Y., Nakajima H., Midorikawa K., Homma-Takeda S., Shimojo N.: Chem. Res. Toxicol. 11, 608
(1998).
3. Garner A.P., Paine M.J.I., Rodriguez-Crespo I., Chinje
E.C., Ortiz de Montellano P., Stratford I.J., Tew D.G.,
Wolf C.R:. Cancer Res. 59, 1929 (1999).
4. Desai A., Stadler W.M., Vogelzang N.J.: Urology 58,
1016 (2001).
5. Camus P., Coudert B., D’Athis P., Foucher P., Delchambre J., Dupront A., Jeannin L. J.: Pharmacol. Exp.
Ther. 259, 1247 (1991).
6. Gram T.E.: Pharmacol. Rev. 49, 297 (1997).
7. Ask K., Heydel J.-H., Piton A., Foucher P., Bonniaud P.,
Camus P.: Nitric Oxide Biol. Chem. 4, 316 (2000)
WP41 DYNAMICS OF CYP2D6
ACTIVITY AND CLINICAL
RESPONSE TO PAROXETINE,
ALPRAZOLAM AND THEIR
COMBINATION IN PATIENTS
WITH DEPRESSIVE DISORDER
EVA HADA·OVÁa, ALEXANDRA ÎOURKOVÁb,
JOSEF TOMANDLc, and BARBARA RAVâUKOVÁd
a
Departments of aPharmacology, bPsychiatry and cBiochemistry, Faculty of Medicine, Masaryk University, Jo‰tova 10,
Brno, dDepartment of Medical Genetics, Children Hospital,
âernopolní 9,Brno, Czech Republic, [email protected]
Numerous antidepressants including tricyclic drugs and
SSRI used in a long-term treatment of depressive disorder
are substrates of polymorphic cytochrome P450 2D6
(CYP2D6). The therapeutic response and occurrence of adverse reactions to the substrate drugs may widely be dependent on the CYP2D6 activity. Paroxetine is known as
one of the most potent CYP 2D6 inhibitors. Due to the
strong inhibitory effect, particularly expressed during
a long-term therapy, paroxetine is able to evoke phenotypic
conversion to PM phenotype even in individuals with EM
genotype (Hada‰ová E., Îourková A.: 18th Eur. Workshop
on Drug Metabolism, Valencia, 16-20 September, 2002,
Abstract Book, p. 52). Nevertheless, neither the extent of
inhibition and its reversibility nor the factual clinical impact of decreased CYP2D6 capacity has definitely been established yet.
The aims of the study were 1) to characterize the dynamics of the paroxetine-induced CYP2D6 inhibition during
the paroxetine therapy and the level of the enzyme restoration after discontinuation of the drug in patients with depressive disorder; 2) to follow the correlations between the
actual CYP2D6 capacity, patient’s genotype and clinical
response.
The study was performed in three groups of patients
(N=30) treated with alprazolam (group I), paroxetine
(group II), and their combination (group III). All patients
gave their written consent with participation in the study.
S180
Alprazolam was chosen as a comparator drug (positive
control) because of its good antidepressant effect without
a remarkable alteration of the CYP2D6 activity. Patients in
all groups were repeatedly tested for CYP2D6 activity in
the following periods of therapy: at the end of the washout
period preceding the start of therapy, at the end of 6 weeks’
therapy, and, finally, 4 weeks after discontinuation of paroxetine therapy in groups II and III and their change-over to
alprazolam (week 10 of the study).
CYP2D6 genotype was determined by allele-specific
PCR with detection of mutations in exons 3, 4, 5 and deletion in exon 6. Dextromethorphan (DEM, 25 mg p.o.) was
used as a probe drug for CYP2D6 phenotype. Concentration of DEM and its metabolites dextrorphan, methoxymorphinan and hydroxymorphinan were measured in 8 hurine by HPLC, the DEM/DOR metabolic ratio (MR) and
proportional excretion of DEM and its metabolites were
calculated and used as parameters of the actual CYP activity.
The therapeutic effect was assessed by HAMA, COVI
and CGI 1 scales. Side effects were assessed according to
UKU rating scale, sexual disorders using ASEX scale.
Homozygous PM genotype was not found within the
group. Heterozygous mutation in CYP2D6 locus was
found in 7 individuals (23.3 %), 4 patients (13.3 %) had deletion in exon 6 at one allele.
Paroxetine evoked significant inhibition of CYP2D6 or
even „PM phenocopy“ in 8 of 16 (50 %) patients treated
with paroxetine alone or in combination with alprazolam.
CYP2D6 inhibition was more pronounced in EM patients
with heterozygous mutation in the CYP locus. Four weeks
after discontinuation of paroxetine therapy, the enzyme
activity was fully restored in 7 of 8 patients switched to PM
phenocopy.
The therapeutic response was satisfactory in all tested
groups. More rapid onset of paroxetine therapy was observed in PMs than in EMs. There was no significant difference between patients with EM and PM phenotype in the
incidence and severity of adverse effects, but, PMs were
more prone to sexual disorders.
The results of this small study suggest that even a profound inhibition of CYP2D6 in individuals with EM genotype may have low clinical relevance namely in drugs with
higher therapeutic indexes like paroxetine and other SSRI
antidepressants.
Supported by grants IGA MH CR (NF6159-3) and
CEZ: J07/98:141100001.
WP42 INHIBITION OF SPONTANEOUSLY PRODUCED NITRIC OXIDE
IN CONVENTIONAL AND
PERFUSED HEPATOCYTE
CULTURE IMPROVES CELL
FUNCTIONALITY
NIKOLINA CANOVÁ, EVA KMONÍâKOVÁ,
and HASSAN FARGHALI
Institute of Pharmacology, 1st Faculty of Medicine, Charles University, Albertov 4, 12800 Prague, Czech Republic,
[email protected]
Introduction
Hepatocytes in culture synthesize increased levels of
nitric oxide (NO) following isolation as a result of induction of nitric oxide synthase (NOS). The isoform of this enzyme (iNOS) is induced in response to stress that occurs
during the two-step perfusion of the liver by EGTA/collagenase for hepatocyte isolation that results in sheer stress
and structural changes of the hepatocyte architecture in the
liver1.
Goals
The present study was therefore directed to assess the
spontaneous NO production under various culture conditions and compare its influence on functional status of hepatocytes in conventional cell culture and in recently described flat membrane bioreactor (FMB)2, i.e. perfused culture.
Methods
Hepatocytes were isolated from rats using the standard
two-phase collagenase perfusion method and seeded on petri
dishes or into the bioreactor entire space formed by polycarbonate plate and gas-permeable 25-_m membrane. The hepatocytes were cultivated on the single collagen layer as a simple monolayer or between two layers of collagen as sandwich. For simple monolayer, supplemented William’s E medium with 5% bovine fetal serum and 1 mM NH4Cl in absence or presence of cyclosporin A (CsA, 2 µg/ml) was utilized. For sandwich culture, the above-mentioned culture
medium and hormone enriched serum free Hybridoma Medium with prednisolone, glucagon, insulin and NH4Cl were
used throughout the study. The hepatocytes in FMB were
perfused in a constant rate of 4.5 ml/h. Samples were collected in appropriate time intervals and nitrite levels were
measured. Hepatocyte functions were tested by urea synthesis, ALT leakage and CsA biotransformation (HPLC-assessed) during 48 hours.
Results and Discussion
It was found here that perfused hepatocyte culture is
metabolically more efficient when contrasted to the static
conventional cell culture (Table 1). Sandwiching hepatocytes in double layer of collagen improved hepatocytes metabolic competence and reduced NO productivity with consequent preservation of cell integrity in culture. Moreover,
the negative influence of NO on urea production was suppressed by the cultivation of hepatocytes in the culture medium containing prednisolone, which is known to inhibit
iNOS expression. Similarly, the addition of the immunosuppressant CsA decreased the spontaneous NO production and enhanced urea biosynthesis. The effect of CsA on
modulation of urea and spontaneous NO production demonstrated flexibility of the perfused culture in observing
the time course of hepatocyte function status in a non-destructive way. The monitoring of NO levels during various
Tab. 1: The rate of spontaneous NO production, urea synthesis (1 mM NH4Cl), CsA disappearance and ALT leakage to the culture media
(WE + FBS: William’s E medium with serum; HM - FBS: hormone enriched serum free Hybridoma Medium with prednisolone) of the
single-collagen-layer and sandwich conventional hepatocyte culture (SCL-/SW-HC) or flat membrane bioreactor (SCL-/SW-FMB). Data
are expressed as mean ± SEM (n = 3-4).
Metabolite
Nitrite levels
nmol/106cells /hr
µg/106cells/hr
Urea levels
ALT levels
mU/106cell/hr
0-48 hr
0-48 hr
0-48 hr
SCL-HC: WE + FBS
5.10 ± 0.53
1.55 ± 0.03
2.47 ± 0.07
SCL-HC: CsA 2 µg/ml
4.56 ± 0.57
1.74 ± 0.04
2.61 ± 0.07
SCL-FMB: WE + FBS
1.07 ± 0.05
2.59 ± 0.44
2.37 ± 0.30
SCL-FMB: CsA 2 µg/ml
0.60 ± 0.11
2.87 ± 0.59
3.23 ± 0.60
SW-FMB: WE + FBS
0.54 ± 0.01
3.82 ± 0.46
1.33 ± 0.09
SW-FMB: HM - FBS
0.34 ± 0.05
4.67 ± 0.97
2.18 ± 0.14
SW-HC: HM - FBS
0.19 ± 0.01
2.27 ± 0.09
1.86 ± 0.05
Culture conditions
S181
CsA%
initial value
24 hr
48 hr
24 ± 3
19 ±3
16 ± 2
9 ±1
steps of isolation and cultivation suggests that spontaneously produced NO has a negative impact on hepatocyte
metabolic and functional integrity.
Supported by MZ NL 7418/3, GACR 305/00/1129 and
Research Project J13/98:111100002.
P450 has a clear preference for C-H hydroxylation in a protein environment,[5] by contrast HRP prefers epoxidation
even under environmental conditions.
REFERENCES
1. de Visser S. P.; Sharma P. K.; Kumar D.; Shaik S.: In
preparation.
2. Ogliaro F.; de Visser S. P.; Cohen S.; Kaneti J.; Shaik
S.: ChemBioChem. 7, 848 (2001).
3. Ogliaro F.; de Visser S. P.; Shaik S.: J. Inorg. Biochem.
91, 554 (2002).
4. de Visser S. P.; Kumar D.; Sharma P. K.; Shaik S. In
preparation.
5. de Visser S. P.; Ogliaro F.; Sharma P. K.; Shaik, S.: J.
Am. Chem. Soc. 124, 11809 (2002).
1. Dilworth C., Bigot-Lasserre D., Bars R.: Toxicol. in
Vitro 15, 623 (2001).
2. Langsch A., Bader A.: Biotechnol. Bioeng. 76, 115
(2001).
WP43 DIFFERENCES AND
COMPARISONS OF THE
ACTIVE SPECIES (CPD I) OF
CYTOCHROME P450 AND
HORSE RADISH PEROXIDASE
AS REVEALED BY DENSITY
FUNCTIONAL THEORETICAL
STUDIES
REFERENCES
WP44 INHIBITION OF INDUCIBLE
NITRIC OXIDE SYNTHASE
ACTIVITY BY OLTIPRAZ
IN VITRO AND IN ACTIVATED
MICROGLIA CELLS
SAM P. de VISSER, PANKAZ K. SHARMA, DEVESH
KUMAR, and SASON SHAIK
The Hebrew University of Jerusalem, 91904 Jerusalem,
Israel.
The enzymes Cytochrome P450 (P450) and Horse radish peroxidase (HRP) share many common features. The
active species of both enzymes consists of an oxo-iron protoporphyrin system which is linked to the amino acid chain
via the sixth ligand of the iron atom. In P450 this linkage
is a thiolate of a cysteinate residue, whereas in HRP the linkage happens via an imidazole group of a histidine amino
acid. Since, thiolate is an anionic ligand while histidine
a neutral ligand this causes differences in reactivity and
properties of the enzymes. Thus, we have initiated a density functional theoretical study using the unrestricted hybrid density functional method, UB3LYP, to model Cpd I of
HRP1 and compared the results to the ones obtained for
a Cpd I model of P450.2 As will be shown, the differences
mainly originate from the „push“ effect of the thiolate ligand, which raises the porphyrin a2u-orbital significantly
due to interactions with the pσ-orbital on sulfur.3 Furthermore, the mixing is strongly dependent on environmental
effects and results in chameleonic behavior of the active
species. Because, the two active species shows differences
in catalytic properties, we tested the reactivity differences
of Cpd I of HRP4 versus Cpd I of P4505 by modelling the
oxidation reaction of propene, which can result in either
double bond epoxidation or C-H hydroxylation. Although,
S182
LAURA LOWE FURGE, PATRICE R. FIELDS,
WHITNEY E. GOODE, MICHAEL C. TRESSLER,
and REGINA STEVENS-TRUSS
Department of Chemistry, Kalamazoo College, Kalamazoo, MI 49006, USA, [email protected]
The clinically relevant drug oltipraz (OPZ) has previously been shown to be a mechanism-based inhibitor of cytochome P450 1A2 and an inducer of glutathione S-transferases. The current study shows that OPZ is also able to
inhibit •NO formation by purified inducible nitric oxide
synthase (iNOS) but not by purified neuronal nitric oxide
synthase (nNOS) in hemoglobin assays. The inhibition of
iNOS by OPZ is mixed-function with an IC50 of 8 µM. In
murine BV-2 microglial cells, an immortalized cell line
that produces •NO in response to lipopolysaccharide
(LPS), OPZ is able to block the formation of nitrite in LPStreated cells. The IC50 in activated BV-2 cells is ~5 µM. The
inhibitory effect of OPZ on LPS-treated BV-2 cells is not
due to cell toxicity. The concentrations of OPZ used in
these studies are comparable to clinically relevant plasma
concentrations in patients treated with OPZ. Finally, treatment of BV-2 cells with OPZ does not suppress production
of iNOS protein. These results support an additional role of
OPZ as an inhibitor of iNOS.
WP45 ENDOTHERIAL NITRIC OXIDE
SYNTHASE REDUCTASE
DOMAIN IS ACTIVATED
BY CALMODULIN AND
PHOSPHORYLATION
WP46 RECRUITMENT OF
GOVERNING ELEMENTS FOR
ELECTRON TRANSFER IN
NITRIC OXIDE SYNTHASE
FAMILY
YOSHITAKA NISHINO, KEITA YAMAMOTO,
ZHI-WEN GUAN, SHIGENOBU KIMURA,
and TAKASHI IYANAGI
MARIE JÁCHYMOVÁa,b, PAVEL MARTÁSEKb,
SATYA PANDAa, THOMAS M. SHEAa,
LINDA J. ROMANa, and BETTIE SUE MASTERSa
a
The electron transfer reaction of NOS reductase domains is controlled by calmodulin (CaM)1,2. These domains
are structurally similar to other dual flavin enzymes like
cytochrome P450 reductase (CPR). Endothelial nitric
oxide synthase (eNOS) function is also regulated by
Ca/CaM, in part by post-traslational mechanisms including
phosphorylation and protein modifications. In this study,
the reductase domain was expressed in E. coli BL21 cells
using expression vector pCWori+. The purified enzyme
contains the FAD, FMN, NADPH and CaM binding sites.
Redox reactions of the reductase domain (FAD/FMN) were
monitored at 457 nm (derived from oxidized flavins),
596nm (derived from semiquinone form of flavins) and
520 nm (derived from FAD semiquinone) by using stopped-flow and rapid-scan spectrometry methods. (1) Reduction of the oxidized enzyme (FAD-FMN) with NADPH
occurred at least two phases. The both rates of reduction
were accelerated in the presence of Ca/CaM. (2) Reduction
of the air-stable semiquinone (FAD-FMNH.) with NADPH
occurred at least two phases, and both phases were accelerated in the presence of Ca/CaM. and the increase at
520 nm was observed only in the presence of Ca/CaM.
From the data of (1) and (2), we suggest that Ca/CaM accelerates both rates of reduction of FAD and intramolecular
one-electron transfer from FAD and FMN. In addition to
these experiments, we have analyzed the autoxidation
spectra of reduced enzymes, which include the mutants of
Ser 633 Asp and Ser1177Asp. The data suggest that the mimic mutation of phosphorylation sites of eNOS reductase
domain activates one-electron transfer reaction between
FAD and FMN.
REFERENCES
1. Matsuda H., Iyanagi T.: Biochim. Biophys. Acta. 1473,
345 (1999).
2. Guan Z-W., Iyanagi T.: Arch. Biochem. Biophys. 412,
65 (2003).
S183
From the Department of Biochemistry, The University of
Texas Health Science Center, San Antonio, Texas, USA
78229, and bDepartment of Pediatrics and Clinical Biochemistry, School of Medicine I, Charles University, Prague,
Czech Republic
Nitric oxide synthases (NOS) exist as bidomain structures containing both N-terminal heme (oxygenase) and
C-terminal flavoprotein (reductase) domains. The oxygenase domain contains the binding sites for iron protoporphyrin IX, pteridine cofactor (H4B), and the substrate,
L-arginine, and serves as the site for O2 binding and substrate conversion to the oxygenated products. The flavoprotein domain shuttles electron equivalents from NADPH
through FAD, then FMN, to the heme. Due to structural similarities among the NOS heme domains, other differences
among the isoforms were sought to explain the unique catalytic turnovers in the production of NO. Each NOS isoform contains more C-terminal residues (iNOS: 21; nNOS:
33; and eNOS: 42) than NADPH-cytochrome P450 reductase (CYPOR), with which they share approximately 60%
sequence similarity. CYPOR chimeras were constructed by
incorporating the C-terminal tails of iNOS, nNOS, and
eNOS, respectively, to the soluble CYPOR sequence to determine their effects on electron transport activities.
The ability of the CYPOR-soluble and CYPOR chimeras to catalyze the cyt c, DCIP, and ferricyanide reduction
was examined. With the incorporation of residues from Ctermini of the iNOS, nNOS, and eNOS, the reduction of all
acceptors was lower, but NADPH oxidation increased by
approximately 60% for all chimeras as compared to soluble
CYPOR. The decrease in acceptor reduction was directly
proportional to the length of the tail; i.e., cyt c reduction
was decreased 20.2%, 25.8%, and 42.5% with the addition
of the iNOS, nNOS, and eNOS tails, respectively. Stoppedflow spectrophotometry under turnover conditions showed
that flavin reduction is five-fold faster with CYPOR-iNOS
than the other proteins. These studies indicate that the
C-termini of the NOS isoforms influence flavoprotein electron transfer to various electron acceptors.
Supported by NIH Grants GM52419 and HL30050 and
Grant No. AQ-1192 from the Robert A.Welch Foundation
to BSSM. PM was supported by Grant GAUK
10/2002/c from the Czech Republic)
xyguanidines. Their interactions with the active site will be
more deeply investigated by UV/Vis, EPR spectroscopy,
and molecular modelling. Some of these compounds could
be of pharmacological interest.
WP47 MODULATION OF ELECTRON
TRANSFER IN NITRIC OXIDE
SYNTHASE USING NON AMINO
ACID GUANIDINES AND
HYDROXYGUANIDINES
REFERENCES
MAGALI MOREAUa, JEAN-LUC BOUCHERa,
SYLVIE DIJOLSa, YVES FRAPARTa, DAVID
LEFEVREa, DENNIS STUEHRb, and
DANIEL MANSUYa
a
Laboratoire de Chimie et Biochimie Pharmacologiques et
Toxicoclogiques, UMR 8601 CNRS, Université R. Descartes, 45 rue des Saints Pères, 75270 Paris cedex 06, France.
b
Department of Immunology, Lerner Research Institute,
Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH
44195, USA. [email protected]
Nitric oxide, NO, is a key inter- and intramolecular molecule involved in the regulation of vascular tone, neuronal
signaling and host response to infection. The biosynthesis
of NO in mammals is catalyzed by constitutive neuronal
and endothelial NO synthases (nNOS and eNOS) and by
inducible NOS (iNOS) expressed in macrophages following induction by inflammatory mediators. All three NOSs
are heme-thiolate proteins related to cytochrome
P450s that produce NO and citrulline from the two-step
oxidation of L-arginine by NADPH and O2, with intermediate formation of N-omega-hydroxy-L-arginine (NOHA).
Many physiopathological situations are characterized either by an overproduction of NO, by a deficit in NO formation, or by the concomitant release of NO and superoxide,
O2..-, a situation that leads to the formation of the potent
oxidizing agent peroxynitrite (O=N-O-O-). Generation of
peroxynitrite by NOSs is greatly enhanced in the absence
of the cofactor tetrahydrobiopterin (BH4) and/or substrate
L-arginine, two compounds that are key-regulators of the
electron transfer from the reductase to the oxygenase domain of NOSs.
We recently reported that some non amino acid N-alkylguanidines, N-aryl and N-alkyl-N’-hydroxyguanidines
are efficiently oxidized by iNOS with formation of NO in
reactions very similar to the oxidation of L-arginine or
NOHA to L-citrulline and NO1-3. We studied the ability of
some of these new non amino acid guanidines and hydroxyguanidines to modulate the electron transfer in the three
recombinant NOSs. We measured their effects on NADPH
consumption and formation of O2.- using a recently synthesized spin trap BMPO4. Some of these compounds strongly
enhanced the NADPH consumption and O2.- generation by
iNOS whereas others are potent inhibitors of the electron
transfer.
These results show that modulation of the activities of
NOSs (NO versus O=N-O-O- formation) can be achieved
by the use of simple non amino acid guanidines and hydro-
S184
1. Dijols S., Perollier C., Lefevre-Groboillot D., Pethe S.,
Attias R., Boucher J.-L., Stuehr D.J., Mansuy D. J.:
Med. Chem. 44, 3199 (2001).
2. Renodon-Corniere A., Dijols S., Perollier C., LefevreGroboillot D., Boucher J.-L., Attias R., Sari M.-A., Stuehr D.J., Mansuy D. J.: Med. Chem. 45, 944 (2002).
3. Dijols S., Boucher J.-L., Lepoivre M., Lefevre-Groboillot D., Moreau M., Frapart Y., Rekka E., Meade A., Stuehr D.J., Mansuy D.: Biochemistry 41, 9286 (2002).
4. Zhao H., Joseph J., Zhang H., Karoui H., Kalyanaraman
B.: Free Rad. Biol. Med. 31, 599(2001).
WP48 ON THE ROLE OF THE
CYSTEINE LIGAND IN
CYTOCHROMES P450
PATRIK RYDBERG, EMMA SIGFRIDSSON,
and ULF RYDE
Department of theoretical chemistry, Lund University,
P.O.B 124, S-221 00 Lund, Sweden
[email protected]
What is the role of the cysteine ligand for the cytochromes P450 and what makes it different from other heme proteins such as the globins, peroxidases and catalases which
have other axial ligands? To answer these questions we have
made a systematic investigation of how the axial ligand in
heme proteins influences geometry, electronic structure, spin
states, and reaction energies. Using the density functional
B3LYP method and medium-sized basis sets, we have compared models with histidine, histidine with a nearby aspartate, cysteine, and tyrosine (with and without an nearby
arginine). We have studied twelve reactants and intermediates of the reaction cycles of these enzymes, including complexes with H2O, OH-, O2-, CH3OH, O2, H2O2, and HO2- in
various formal oxidation states of the iron (II to V).
The results show that the most pronounced difference is
found between the neutral histidine ligand an the other ligands. For example the histidine complexes have lower reduction potentials and different proton affinities than complexes with the other ligands. In particular, it is harder to
reduce and protonate the FeIIO2 complex with histidine, in
accordance with the O2 carrier function of globins and the
oxidative chemistry involving a FeIIIO2H intermediate of
proteins with the other ligands. For most properties, the
trend cysteine < tyrosine < histidine with aspartate < histidine is found, indicating the donor capacity of the various
ligands. However, the tyrosine complexes have a unusually
low affinity for neutral ligands, giving them a slightly enhanced driving force in the oxidation of H2O2 by compound
I. In vacuum, the formation of compound I is less favourable for histidine than for the other ligands, but this effect
disappears in polar solvents. Likewise, we obtain the trend
tyrosine < histidine < histidine with aspartate < cysteine for
difference in reaction energies of the hetero- and homolytic brekage of the O-O bond.
WP49 IS NOS A NOX-SYNTHASE?
J. SANTOLINIa, Z.-Q. WANGb, D. KONASb,
and D. J. STUEHRb
a
Laboratoire de Biophysique du Stress Oxydant, DBJCSBE - CEA-Saclay 91191 Gif Sur Yvette Cedex, France
b
Department of Immunology Cleveland Clinic Foundation
NB3-120 - 9300, Euclid Avenue 44195, Cleveland OHIO,
USA
NO-Synthases present a novel system of self-regulation. Due to a highly efficient geminate recombination, NO
is trapped in NOS heme pocket instantaneously after biosynthesis. Therefore, the product of NOS catalysis is not
NO but a heme-NO complex. Based on this approach, we
recently unified the 3 NOS isoforms in a single kinetic model that explained their divergent catalytic behaviors1: Our
model is based on the coexistence of two regenerative
pathways linked to the nature of the heme-NO complex:
i) FeIII-NO gives rise to a rapid productive cycle that releases NO and ii) FeII-NO leads NOS into a slow and futile
cycle that releases higher N-oxides2. Partitioning between
these two pathways, modifies the catalytic rate, the nature
of the catalytic product and the overall efficiency of all three NOSs. Two kinetic steps mainly control the whole process:
FeII-NO oxidation
We characterized the relationship between heme redox
potential and FeII-NO reactivity by measuring the heme
midpoint potential and the rate of FeII-NO oxidation for
iNOS, nNOS, and mutants containing combinations of pteridines and substrate analogs to generate a wide range of
potentials. Globally the rate of FeII-NO oxidation is controlled by heme redox status; heme potential lowering
seems to favor a FeIII-NO- like behavior and to increase
heme-NO reactivity with O23. Our results also highlight
a complex correlation between this reactivity and electronic back-donation from the proximal thiolate bond.
Ferric heme reduction
In order to understand the reasons of the variability of
heme reduction within the three major mammalian NOS
isoforms, we determined the midpoint potential of flavin
S185
redox couples of their reductase domains4. iNOS
FMNH2/FMNH° potential is significantly higher than its
heme midpoint potential. Consequently, for iNOS, the
electron transfer to the oxygenase domain is controlled by
the heme potential increase observed upon substrate binding. The 40-fold difference in heme reduction rate of
nNOS and eNOS appears to be linked to modifications of
their FAD/FMN redox couples and changes in hydride
transfer rate and electron cycling within the reductase. Based on these results, we proposed a model of electron shuttling within NOS and an explanation for the specific oxygen
control of heme reduction in eNOS.
Tuning of the two key kinetic parameters allows a given NOS to broadly modify its catalysis as a function of its
physiologic environment. It particularly decides of the balance between NO-releasing complex (FeIII-NO) and NOxreleasing species (FeII-NO). We are currently trying to determine the extrinsic and intrinsic parameters that determine the nature of the synthesized N-oxide.
REFERENCES
1. Santolini J., Adak S., Curran C. M., Stuehr, D. J.:
J. Biol. Chem. 276, 1233 (2001).
2. Santolini J., Meade A., Stuehr D. J.: J. Biol. Chem. 276,
48887 (2001).
3. Santolini J., Wang Z.-Q., Meade A.L., Panda K., Stankovich M., Stuehr D.J.: Submitted to Biochemistry
4. Santolini J., Meade A.L., Stephens A.W., Stankovich,
M., Stuehr D.J. Submitted to Biochemistry
WP50 INDUCTION AND MODULATION
OF INDUCIBLE NITRIC OXIDE
SYNTHASE EXPRESSION
FOLLOWING KERATOPLASTY
IN MICE
PETRA ST¤E·TÍKOVÁa,b, JARMILA PL·KOVÁb,
JIND¤ICH MARTÍNEKc, MARTIN FILIPECb, and
HASSAN FARGHALIa
a
Institute of Pharmacology, Albertov 4, bDepartment of
Ophthalmology, U nemocnice 2, cInstitute of Histology and
Embryology, Albertov 4, 1st Faculty of Medicine, Charles
University, Prague 2, Czech Republic, [email protected]
The inducible nitric oxide synthase (iNOS) is known to
be expressed following exposure to cytokines or bacterial
lipopolysaccharides. It has been suggested that nitric oxide
(NO), in particular that which is generated via the induction of iNOS, is an important contributor to inflammatory
processes and allograft rejection. Immunohistological studies showed a massive infiltration of rejected corneal allografts by macrophages and CD4+ lymphocytes.1 CD8+
lymphocytes, activated by CD4+ T-cells, have been considered for a long time to be the main effector cells in the rejection process. Further studies brought evidence that activated macrophages are the key cytotoxic cell population in
process of skin allograft rejection in mice.2 The present
study was directed to investigate iNOS expression following orthotopic allogeneic corneal transplantation in mice
(C57BL/10; H-2b to BALB/c; H-2d) and to apply such
a strategy of modulation of iNOS expression that will lead
to the prolongation of corneal allograft survival. The expression of the gene for iNOS was assessed in non-rejected
and rejected corneas using RT-PCR. The iNOS protein was
detected by means of immunohistological techniques in
crysections stained with anti-iNOS polyclonal antibody.
4,5-diaminofluorescein diacetate (DAF-2 DA) enables
a direct detection of NO in tissues. Our results suggest that
iNOS mRNA and iNOS protein are expressed during rejection of corneal allograft, whereas non-rejected transplants and non-transplanted corneas do not exert any expression of iNOS. Using DAF-2 DA, higher fluorescence
was found in rejected grafts than in non-rejected grafts.
The application of the selective immunosuppressant FK
506 (0.3 mg/kg/per day) or the specific inhibitor of iNOS
aminoguanidine (100 mg/kg/per day) prevents corneal
allograft rejection within 4 weeks in 71% and 57% of animals, respectively, (compared to 29% of clear grafts in
controls) and suppresses iNOS at different levels. In conclusion, iNOS was strongly expressed in rejecting grafts of
the control group and the suppression of iNOS prolonged
the corneal allograft survival, under the present conditions.
Acknowledgments
This study was supported by grants IGA MZ NL/7418-3
and NI/7531-3, and research projects MSM 111100002 and
111100005. FK 506 was kindly supplied by Fujisawa
GmbH (Munich, Germany).
REFERENCES
1. Kuffová L., Veselá V., Filipec M., HoláÀ V., Lumsden
L., Forrester J. V.: Br. J. Ophthalmol. 83, 1364, (1999).
2. Yamamoto N., Einaga-Naito K., Kuriyama M., Kawada
Y., Yoshida R.: Transplantation 65, 818, (1998).
WP51 MECHANISTIC STUDIES ON
THE INTRAMOLECULAR
ONE-ELECTRON TRANSFER
BETWEEN THE TWO FLAVINS
IN INOS FLAVIN DOMAIN
AND NADPH-CYTOCHROME
P450 REDUCTASE
KEITA YAMAMOTO, ZHI-WEN GUAN,
YOSHITAKA NISHINO, SHIGENOBU KIMURA,
and TAKASHI IYANAGI
Flavin electron transferases, including NOSs and
NADPH-Cytochrome P450 reductase (CPR) catalyze oneelectron reduction of quinones1,2. In this work, the inducible nitric-oxide synthase (iNOS) reductase domain was
co-expressed with CaM. The purified reductase domain catayzed aerobic NADPH-oxidation in the presence of the
model quinone compound menadione (MD). The MD-mediated NADPH oxidation was inhibited in the presence of
NAD(P)H:quinone oxidoreductase(QR). The one-electron
reduction potential of various Qs was related to their reduction rates by both the iNOS reductase domain and CPR.
In both iNOS reductase domain and CPR, the semiquinone
species (FAD-FMNH.) were major intermediates observed
during the oxidation of the reduced enzyme by MD, but the
fully reduced flavin species did not significantly accumulate under these conditions. Air-stable semiquinone form
(FAD-FMNH.) and the FAD semiquinone (FADH.) do not
react rapidly with MD, but the fully reduced species of
both flavins, FAD and FMN, can donate rapidly one electron to MD. In the addition to air-stable semiquinone intermediate, the disemiquinone species (FADH.-FMNH.) as an
intermediate was observed in the iNOS reductase domain.
Finally, the iNOS reductase domain and CPR can shuttle
between the 1e/3e level during one-electron reduction of
MD. The CaM-bound state of the iNOS flavin domain showed similar rdox properties to those of CPR. Furthermore,
the present data indicate that the low level of reactivity
with electron acceptors of both FAD and FMN semiquinones could enable a successive one-electron transfer from
the reduced FMN (FMNH2) to Cytochrome P450.
REFERENCES
1. Iyanagi T., Yamazaki I.: Biochim. Biophys. Acta. 216,
282 (1970).
2. Matsuda H., Kimura S., Iyanagi T.: Biochim. Biophys.
Acta. 1459, 106 (2000).
S186
WP52 XANTHATES
(DITHIOCARBONATES)
METABOLISM BY SOME
MONOOXYGENASES
WP53 THE METABOLIC RATIO
CARBAMAZEPINE/CARBAMAZEPINE-EPOXIDE IN CHILDREN
AND ADULT PATIENTS ON
ANTIEPILEPTIC POLYTHERAPY
STANISLAV YANEVa, BOZHIDARKA PANDOVAa,
TOMMASO A. VANNELLIb, VILLIANA TODOROVAa,
and PAUL R. ORTIZ DE MONTELLANOb
BRANKA BRZAKOVICa, MILENA POKRAJACa,
ÎARKO MARTINOVICb, BRANISLAVA MILJKOVICa,
and RUZICA VELICKOVICb
a
Dept. Drug Toxicology, Inst. Physiology, Bulgarian Academy of Sciences, Sofia, Bulgaria, bDepartment of Pharmaceutical Chemistry, University of California, San Francisco, CA,
USA
Xanthates (salts of alkyl or aryl derivatives of dithiocarbonic acid, ROCS2K) are well known metal chelating
agents. In different chemical and biological systems they
behave also as very potent one electron acceptors. By pyrolytic reaction at 300 oC they decomposed to pure olefins.
Our studies have shown that such pyrolytic cleavage of
xanthate molecule could be reproduced at 37 oC by any enzymatic or nonenzymatic system which generates active
oxygen species as hydroxyl radicals (Fe/EDTA/H2O2, xanthine-xanthine oxidase, hemoglobin, cytochrome P450
(CYP)). The primary changes in xanthate molecule after
CYP attack is one or two hydrogens abstraction from the
first carbon atom of the alkyl chain. The resulting intermediate(s) irreversibly bound to enzyme protein. Such metabolic transformation was exert only by CYP 2B1/2B6
(with high affinity) and CYP 2E1 (with lower affinity). All
others CYP are ineffective or only inhibited by xanthates.
By this way xanthates behaved as potent and selective mechanism-based inactivators of some CYP enzymes.
3D QSAR studies showed that CYP xanthates inhibitory
potency depends mainly by the derivative lipid solubility
and molecular volume as the inactivation potency (rate of
xanthate metabolism by CYP) depends mainly by the
charge of the first carbon atom of the alkyl chain.
Differently from CYP, xanthates are oxidized by some
FMO’s at the sulfur part of the molecule to the corresponding S-peroxido dithiocarbonate derivatives (perxanthates). The same sulfur oxidation occurs in pure chemical
system containing hydrogen peroxide.
The readiness of xanthate molecule to interact with different reactive oxygen species reflect in from one side their
potent antioxidant and scavenger activity, and from the other side can explain the differences in their metabolic profile after attack from different monooxygenases. Knowing
that the balance between the oxygen species in the cell and
other active intermediates like NO, is playing crucial role
in cell homeostasis, such xanthates properties could be on
the background of their antiviral and anticancer activity
triggered by the unusual xanthate metabolism in the cell.
S187
a
Department of Pharmacokinetics, Faculty of Pharmacy,
University of Belgrade,
b
Institute for Mental Health, Belgrade, Serbia and Montenegro, [email protected]
Carbamazepine (CBZ) is considered a first-line drug in
the treatment of most forms of epilepsy. Its pharmacokinetics shows both inter- and intraindividual variability with
respect to age, dose, concomitant therapy and disease. The
major metabolic pathway is the epoxide-diol enzymatic
formation and carbamazepine-10,11- epoxide (CBZE) is an
active metabolite with similar effects as the parent drug 1,2.
In this study we investigated metabolic ratios of CBZ
/CBZE in children and adult patients on CBZ-antiepileptic
polytherapy, as well as compared other pharmacokinetic
parameters between the two groups.
Patients with epilepsy receiving CBZ in combination
with other antiepileptic drugs (AEDs) were studied. AEDs
taken in addition to CBZ included phenobarbital, valproic
acid, lamotrigin or clonazepam. Thirty two patients: 21
children, 11 ± 2 years, mean ± SD and 11 adults, 31 ± 4 years, mean ± SD, were taken steady-state heparinized blood
samples at 8 am, and at 1 and 6 pm. CBZ and CBZE were
measured by UV-reversed-phase high-performance liquid
chromatographic (RP-HPLC) method, after their extraction
from plasma with ethyl acetate.
Pharmacokinetic parameters: metabolic ratio of parent
drug and its metabolite (CBZ/CBZE), concentration-dose
Tab. 1. Comparison of pharmacokinetic parameters of CBZ between children and adult patients with epilepsy
Parameter
DCBZ (mg/kg/day)
Group
Significance
Children
Adults
17,23 ± 5,36
17,32 ± 5,59
NS
CCBZ (mg/L)
6,82 ± 3,24
8,60 ± 2,60
p < 0,05
CCBZE (mg/L)
1,32 ± 0,80
1,37 ± 0,83
NS
CCBZ
CCBZE
5,99 ± 2,92
10,45 ± 10,35
p < 0,05
CCBZ (mg/L)
D mg/kg/day
0,41 ± 0,20
0,66 ± 0,56
p < 0,05
CLCBZ (L/h/kg)
0,347 ± 0,246
0,292 ± 0,131
NS
AUCCBZ (mgh/L)
30,56 ± 30,26
75,40 ± 41,58
NS
ratio for CBZ (C/D), clearance (CL) as a direct measure of
the activity of metabolizing enzymes, and area under the
curve (AUC), were compared between the adults and children using Mann-Whitney test3. Summary results are presented in Table 1.
Diferences in CBZ/CBZE metabolic ratios between
children and adults on equivalent antiepileptic polytherapy
obtained in the study were dependent on differences in
CBZ plasma concentrations between the two groups of patients. Metabolite, CBZE plasma levels, as well as plasma
clearance values for CBZ, were comparable.
REFERENCES
1. Bertilsson L., Tomson T.: Clin. Pharmacokinet. 11, 177
(1986).
2. Svinarov D.A., Pippeneger C.E.: Ther. Drug Monitor.
18, 660 (1996).
3. Liu H., Delgado M.R.: Ther. Drug Monitor. 16, 132
(1994).
mes, with possible consequences for the effects of metabolically inactivated paclitaxel in these animal species. It became obvious that despite various similarities between human and pig metabolism, the profile of paclitaxel metabolites in the studied species is different and 6α-OHP remains uniquely human metabolite. In the study of paclitaxel
metabolism by different CYP cDNA-expressed enzymes
(CYP1A2, 1B1, 2A6, 2C9, 2E1 and 3A4), only CYP3A4
enzyme formed C3’-OHP, C2-OHP and one unknown metabolite.
The roles of CYP3A and CYP2C8 in the formation of
paclitaxel metabolites were investigated using specific inhibitors: troleandomycin (inhibitor of CYP3A4) and fisetin, which we found to inhibit CYP2C8 activity1.
CYP3A inhibitor troleandomycin inhibited the formation
of C3’-OHP, C2-OHP, di-OHP as well as the unknown rat,
minipig and pig OHP. Fisetin strongly inhibited the formation of 6α-OHP in human microsomes, which is catalyzed
by human unique CYP2C8 enzyme.
Acknowledgements
This work was supported by grant IGA NL/6715-3. The
authors are indebted to Dr. ·imek (AV CR, âeské Budûjovice) for the identification of paclitaxel metabolites by
MS/MS
WP54 NEW IN VITRO METABOLITES
OF PACLITAXEL IN HUMANS,
RATS, MINIPIGS AND REGULAR
PIGS AND CYP INVOLVED
IN THEIR FORMATION
REFERENCES:
1. Gut I., Václavíková R., Horsk˘ S., Ozgová ·., Souãek
P., Sapporo, Japan, July 22-26, 2002, Abs. P-26-C-41,
http://www.convention.co.jp.MDO2002
RADKA VÁCLAVÍKOVÁ, STANISLAV HORSK¯, and
IVAN GUT
National Institute of Public Health, ·robárova 48, 100 42
Prague 10, Czech Republic, [email protected]
Paclitaxel is an important recently introduced antineoplastic drug. We investigated cytochrome P450 (CYP) - cytalyzed metabolism of paclitaxel in rat, minipig, regular
pig and human liver microsomes. In the rat microsomes,
paclitaxel was metabolized mainly to C3’-hydroxypaclitaxel (C3’-OHP), less to C2-hydroxypaclitaxel (C2-OHP),
di-hydroxypaclitaxel (di-OHP) and another so far unknown
monohydroxylated paclitaxel. In the minipig microsomes,
this unknown hydroxypaclitaxel was the main metabolite,
whereas C3’-OHP and C2-OHP were minor products. In
the regular pig microsomes, the same metabolic pattern
was demonstrated. In human liver microsomes 6α-hydroxypaclitaxel (6α-OHP) was the main metabolite, followed
by C3’-OHP, C2-OHP and two other metabolites not yet
fully characterized. However, the proportion of 6α-OHP
and C3’-OHP in different human microsomes varied significantly. Kinetic parametres (Km and Vmax) of production of
various metabolites in rat, minipig, pig and human liver
microsomes revealed species and individual differences.
The human microsomes oxidized paclitaxel at substantially
higher rates than the rat, minipig and regular pig microso-
S188
WP55 QUANTITATIVE EVALUATION
OF RELATIVE CONTRIBUTION
OF HUMAN CYTOCHROME
P-450 ISOENZYMES (CYPS) TO
THE METABOLISM OF
PROMAZINE
JACEK WÓJCIKOWSKIa,
W¸ADYS¸AWA ANNA DANIELa,
LYDIANE PICHARD-GARCIAb, and
PATRICK MAURELb
a
Institute of Pharmacology, Polish Academy of Sciences,
Sm´tna 12, 31-343 Kraków, Poland; bInstitut National de
la Santé et de la Recherche M(dicale U128, 1919 Route de
Mende, 34293 Montpellier, France.
Our previous investigations indicated that the catalysis
of promazine in humans do not exhibit a strict CYP isoform preference. The present detailed experiments performed at therapeutic concentrations of the drug were aimed
to estimate quantitively relative contribution of CYPs to
promazine 5-sulfoxidation and N-demethylation. The
amount of neuroleptic metabolites formed in liver microsomes, cDNA-expressed human CYPs or hepatocytes was
assayed using HPLC with UV detection.
In human liver microsomes, the formation of promazine 5-sulphoxide and N-desmethylpromazine was significantly correlated with the level of CYP1A2 and ethoxyresorufin O-deethylase and acetanilide 4-hydroxylase activities, as well as with the level of CYP3A4 and cyclosporin
A oxidase activity. Moreover, the formation of N-desmethylpromazine was correlated well with S-mephenytoin
4’-hydroxylation. Furafylline (a CYP1A2 inhibitor) and
ketoconazole (a CYP3A4 inhibitor) significantly decreased
the rate of promazine 5-sulphoxidation, while furafylline
and ticlopidine (a CYP2C19 inhibitor) significantly decreased the rate of promazine N-demethylation in human liver
microsomes. The cDNA-expressed human CYPs (1A1,
1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4) generated different amounts of promazine metabolites. The kinetic parameters (Lineweaver-Burk analysis) obtained in cDNA-expressed human CYPs showed distinct inter-isoform differences (Km = 37 - 350 µM, Vmax = 0.15 - 2.47 pmo/pmol
CYP isoform/min in the case of 5-sulphoxidation and Km =
47 - 741 µM, Vmax = 0.33 - 13.70 pmo/pmol CYP isoform/min in the case of N-demethylation), which was consistent with the multienzyme Eadie-Hofstee plots derived
from liver microsomes. The highest intrinsic clearance
(Vmax/Km) was found for CYP1A subfamily, CYP3A4 and
CYP2B6 in the case of 5-sulphoxidation, and for
CYP2C19, CYP1A subfamily and CYP2B6 in the case of
N-demethylation. In a primary culture of human hepatocytes, TCDD (a CYP1A subfamily inducer), as well as rifampicin (mainly a CYP3A4 inducer) induced the formation of promazine 5-sulphoxide and N-desmethylpromazine.
Regarding the relative expression of various CYPs in
human liver, the obtained results indicate that CYP1A2
(31%) and CYP3A4 (39%) are the main isoforms responsible for 5-sulphoxidation, while CYP1A2 (35%) and
CYP2C19 (32%) are the basic isoforms that catalyze N-demethylation of promazine in human liver. Of the other isoforms studied, CYP2C9 (14%) and CYP3A4 (13%) contribute to a lesser degree to promazine 5-sulphoxidation and
N-demethylation, respectively. The role of CYP2A6,
CYP2B6, CYP2D6 and CYP2E1 in the investigated metabolic pathways of promazine seems negligible.
Since phenothiazine neuroleptics are combined with
antidepressants or carbamazepine in the treatment of complex or „treatment-resistant“ psychiatric disorders, the obtained results may have significant implications for the
prediction of potential drug-drug interactions involving
promazine.
S189
WP56 STIMULATION OF HUMAN
CYTOCHROME P450 3A4
ACTIVITY BY ANIONIC
PHOSPHOLIPIDS AND THEIR
MICRO-DOMAINS
KEON-HEE KIMa, TAEHO AHNb, and
CHUL-HO YUNa
a
Division of Life Science, Pai-Chai University, Taejon 302735, Republic of Korea, bDepartment of Biochemistry, College
of Veterinary Medicine, Chonnam National University,
Kwangju 500-757, Republic of Korea, [email protected]
In humans, cytochrome P450 (CYP) 3A4 is the major
enzyme expressed in liver and plays a major role in the metabolism of many drugs and procarcinogens.1 CYP3A4
seems to have a large cytoplasmic domain anchored to the
endoplasmic reticulum membrane by an amino-terminal
transmembrane segment.2 We has shown that the activities
and conformations of rabbit CYP1A23,4 and CYP2B15 are
modulated by phospholipids. It is also known that the composition of phospholipids in liver microsomes is dependent
on physiological conditions.6 Role of phospholipids in the
catalytic activity and membrane binding of CYP3A4 was
investigated with model membranes. When phosphatidylcholine (PC) matrix was replaced with anionic phospholipids [phosphatidic acid (PA), phosphatidylserine (PS), and
phosphatidylinositol] and phosphatidylethanolamine (PE),
all anionic phospholipids increased the activity. PA was the
most efficient and the enhancement reached 15-fold in the
presence of 50% PA and 50% PC compared with that of
100% PC. Binding and insertion of CYP3A4 to the membranes was also increased by anionic phospholipids and PA
exerted the most efficient effect. PE was found to act as an
inert matrix phospholipid, similar to PC in PC/PE binary
system without anionic phospholipids, as it showed very
little effect on the catalytic activity of CYP3A4 and the
binding of CYP3A4 to membranes. However, in ternary
system of PC/PE/PA or PC/PE/PS with a fixed concentration of 10 or 20 mol% anionic phospholipid, the catalytic
activity further increased as a function of PE concentration.
From the observations obtained from the excimer fluorescence of pyrene-PA and NBD-PA and the resonance energy
transfer between pyrene-PA and BODIPY-PA, it was deduced that PE caused PA-enriched domains in PC/PE/PA
membranes like the case of PC/PE/PS previously suggested.7 In ternary system of PC/PE/PA or PC/PE/PS, the activity further increased as a function of PE concentration. PE
was found to cause PA- or PS-enriched domains in the ternary system. The phospholipid-dependent activity change
coincides with conformational change including altered
Trp fluorescence and CD spectra in visible region of
CYP3A4. These results suggest that the formation of
micro-domains enriched with anionic phospholipid is important for the initial binding to membranes and the cataly-
tic activity of CYP3A4. [This work was supported by Korea Research Foundation Grant (KRF-2000-015-FS0002].
REFERENCES
1. Guengerich F.P.: Chem. Res. Toxicol. 14, 611 (2001).
2. Black S.D.: FASEB J. 6, 680 (1992).
3. Yun C.-H., Song M., Kim H.: J. Biol. Chem. 272, 19725
(1997).
4. Ahn T., Guengerich F.P., Yun C.-H.: Biochemistry 37,
12860 (1998).
5. Yun C.-H., Ahn, T., Guengerich F.P.: Arch. Biochem.
Biophys. 356, 229 (1998).
6. DePierre J., Dallner G.: Biochim. Biophys. Acta 300, 1
(1973).
1.9 µM, respectively. Hemin also inhibited reduction of cytochrome c and ferricyanide by NPR up to 45%. Spectrally
detectable CYP was destroyed in human liver microsomes
and purified CYP in a reconstituted system in the presence
of hemin and an NADPH-generating system. These results
suggest the inhibitory effect of hemin on the CYP-catalyzed reactions may come from the inability of an efficient
electron transfer from NPR to CYP. It also can be suggested that the antimutagenic effect of hemin might be due to
inhibition of CYP and NPR enzyme, involved in the bioactivation of mutagens by hemin. [This work was supported
by Korea Research Foundation Grant (KRF-2000-015FS0002)].
REFERENCES
1. Osawa Y., Yarborough C., Osawa Y.: Science 215, 1249
(1982).
2. Stickney D.R., Anderson L.D., Slater J.B., Ahlem C.N.,
Kirk G.A., Schweighardt S.A., Frincke J.M.: Cancer
Res. 51, 6650 (1991).
3. Furman G.M., Silverman D.M., Schatz R.A.: Toxicology 68, 75 (1991).
4. Yoshida T., Migita C.T.: J. Inorg. Biochem. 82,
33(2000).
5. Katoh Y., Nemoto N., Tanaka M., Takayama S.: Mutat.
Res. 121, 153 (1983).
WP57 POTENT INHIBITION OF
HUMAN CYTOCHROME
P450-CATALYZED REACTIONS
BY HEMIN
EUN-YOUNG KIMa, JOON-SIK KIMb,
MIN-YOUNG KIMb, WOO-SUK KOHc,
F. PETER GUENGERICHd, and CHUL-HO YUNa
a
Division of Life Science and bAngioLab, Pai-Chai University, Taejon 302-735, Republic of Korea, cToxicology Research Center, Korea Research Institute of Chemical Technology, Taejon 305-606, Republic of Korea, dDepartment of
Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN 372320146, USA, [email protected]
An inhibition or destruction of CYP may cause prolongation of xenobiotic action, increase of decrease in xenobiotic toxicity, and even affect physiological functions, e. g.
steroidal biosynthesis.1 Selective destruction of various
CYP types alters the activation/detoxication ratio of procarcinogens and thus may increase the cancer risk in conditions of combined exposure.2,3 Heme taken up by hepatocytes is known to be oxidized to biliverdin by the microsomal heme oxygenase system which consists of heme oxygenase and NADPH-cytochrome P450 reductase (NPR).4
Hemin, a stable form of heme, has an antimutagenic effect.5 Inhibitory effects of hemin on the cytochrome P450
(CYP)-catalyzed reactions of human liver microsomes and
reconstituted systems containing purified CYP and NPR
were seen. Hemin non-specifically inhibited all of the microsomal CYP activities observed. Hemin also inhibited
the reactions of 7-ethoxy-4-(trifluoromethyl)coumarin
O-deethylation, 3-[2-(N,N-diethyl-Nmethylammonium)ethyl]-7-methoxy-4-methylcoumarin O-deethylation, and
benzo(α)pyrene 3-hydroxylation, catalyzed by purified
CYPs 1A2, 2D6, and 3A4, with IC50 values of 25, 17, and
S190
WP58 GENETIC POLYMORPHISMS IN
HUMAN CYP2A6 GENE AND
INTERINDIVIDUAL
DIFFERENCES IN NICOTINE
METABOLISM
MIKI NAKAJIMA, RYOKO YOSHIDA,
KIYOKO NISHIMURA, JUN-TACK KWON,
and TSUYOSHI YOKOI
Division of Drug Metabolism, Faculty of Pharmaceutical
Sciences, Kanazawa University, Takara-machi 13-1, Kanazawa, Japan, [email protected]
CYP2A6 catalyzes nicotine C-oxidation leading to cotinine formation, a major metabolic pathway of nicotine in
humans. There are genetic polymorphisms in the human
CYP2A6 gene. We determined the phenotyping of in vivo
nicotine metabolism and the genotyping of the CYP2A6
gene in 92 Japanese and 209 Koreans1,2. Among them, 3 Japanese and 4 Korean subjects who were absolutely deficient in cotinine formation were genotyped as CYP2A6*4
(whole deletion)/CYP2A6*4. The other poor metabolizers
whose nicotine metabolism was impaired were genotyped
as CYP2A6*7(I471T)/CYP2A6*4 or CYP2A6*10 (I471T,
R485L)/CYP2A6*4, indicating that the CYP2A6 enzyma-
tic activity is lost in the subjects homozygous for either
CYP2A6*4, CYP2A6*7, or CYP2A6*10, or heterozygous
for these alleles in combination3. The CYP2A6*1X2 (duplication) allele was found in only one Korean subject (0.5%)
whose nicotine metabolic potency was not very high.
An allele possessing a point mutation in the TATA box
termed CYP2A6*9 (T-48G) has been reported to decrease
the transcriptional activity in vitro as assessed by luciferase
assay4. We investigated the effects of the CYP2A6*9 allele
on the in vivo enzymatic activity by evaluating nicotine
metabolism. Furthermore, the effects on the expression level of CYP2A6 mRNA and coumarin 7-hydroxylase activities in human livers in vitro were also investigated. The
mutation of T-48G was found only on the CYP2A6*1A allele but not on the CYP2A6*1B allele. In the in vivo study,
the allele frequencies of CYP2A6*9 in Japanese (n = 92)
and Koreans (n = 209) were 21.3% and 22.3%, respectively. In both populations, the cotinine/nicotine ratio as
an index of nicotine metabolism could be put in the order
of subjects genotyped as CYP2A6*1A/CYP2A6*1A >
CYP2A6*1A/CYP2A6*9 > CYP2A6*9/CYP2A6*9. In the
in vitro study, the expression levels of CYP2A6 mRNA in
human livers and coumarin 7-hydroxylase activity in human liver microsomes that were genotyped as
CYP2A6*1/CYP2A6*9 and CYP2A6*9/CYP2A6*9 were lower than those in human livers genotyped as
CYP2A6*1/CYP2A6*1. Thus, the mutation in TATA box
(CYP2A6*9 allele) caused the decreased expression level
of CYP2A6 mRNA and enzymatic activity in vivo and in
vitro.
REFERENCES
1. Nakajima M., Kwon J.-T., Tanaka N., Zenta T., Yamamoto Y., Yamamoto H., Yamazaki H., Yamamoto T.,
Kuroiwa Y., Yokoi T.: Clin. Pharmacol. Ther. 69, 72
(2001).
2. Kwon J.-T., Nakajima M., Chai S., Yom Y.-K., Kim H.K., Yamazaki H., Sohn D.-R., Yamamoto T., Kuroiwa
Y., Yokoi T.: Pharmacogenetics 11, 317 (2001).
3. Yoshida R., Nakajima M., Watanabe Y., Kwon J.T., Yokoi T.: Br. J. Clin. Pharmacol. 54, 511 (2002).
4. Pitarque M., von Richter O., Oke B., Berkkan H.,
Oscarson M., Ingelman-Sundberg M.: Biochem. Biophys. Res. Commun. 284, 455 (2001).
S191
WP59 GENETIC POLYMORPHISM OF
CANINE CYTOCHROME P450
2D15 GENE
IOANNIS S. PAPPAS, and DIMITRIOS S. KATSIABAS
Laboratory of Veterinary Pharmacology and Toxicology,
Faculty of Veterinary Medicine, University of Thessaly,
Trikalon 224, 43100 Karditsa, Greece, [email protected]
The human polymorphic isoenzyme CYP2D6 has a major role in the oxidative metabolism of many drugs1. The
corresponding canine CYP2D15 was found to metabolize
the COX-2 inhibitor, celecoxib, with different velocities
suggesting that the gene may be polymorphic2. In this
study, we have investigated the polymorphism of canine
CYP2D15 that it is cloned using conventional PCR techniques from exon5 to the end of exon7 and sequenced. DNA
was isolated from whole blood samples of 51 mixed breed
dogs from the local animal hospital, amplified using PCR
and digested with restriction enzymes.
PCR and RFLP conditions: a PCR reaction was performed for each DNA sample in a total volume of 50 µl using
CYP2D15 primers (CYP2D15ex5-F: 5’-GCCCTGAACTCCATCCCCGTG-3’, CYP2D15Aex7-B: 5’-CTTGGGGATGAGGAAGCCCTG-3’) and 0,5 units Platinum Taq DNA
polymerase. The PCR conditions consisted of an initial
melting temperature 95 oC (2.5 min) followed by 35 cycles
of melting (95 oC, 0.5 min), annealing (60 oC, 1 min),
and extension (72 oC 1,5 min) with a final extension step
(72 oC, 10 min). To determine the polymorphism of
CYP2D15, the amplified DNA (872 bp) was digested with
1 unit of each
restriction enzymes, separately, at 37 oC
for 3 h:
a) StuI that
cuts amplified
DNA when it
is
mutated
into 798 bp
and 74 bp.
When DNA
is not mutated StuI do
not cut, and
b) DrdI that cuts amplified DNA sequences when it is mutated into three fragments of 625 bp, 211 bp and 36 bp and
when it is not the 625 bp DNA fragment upon digestion gave
two bands of 397 and 228 bp. Digestion products were then
analyzed electrophoretically on an ethidium bromide-stained
2% agarose gel at 100 V for 90 min (Fig. 1).
Alignment of the cloned ex5-ex7 DNA sequence with the
known canine CYP2D15 revealed that there are two polymorphic sites in the DNA coding region: 1) A834T (CYP2D15*1)
located in exon 5, and 2) A1005G (CYP2D15*2) located in
exon 6 (numbered according to canine CYP2D15 cDNA with
accession number D17397)3. These polymorphic sites have
as result the amino acid change of Ile250➔Phe and
Ile307➔Val. The Phe250 allele (T834) occurred at a frequency of 0.275 (homozygotes for the SNP) and none homozygote for A834 was found. Heterozygotes was occurred at
a frequency of 0.725. For the other polymorphic site
(A1005G), the Val307 allele (G1005) occurred at a frequency
of 0.843 (homozygotes for the SNP) and none homozygote
for A1005 was found. Heterozygotes was occurred at a frequency of 0.157. These preliminary results suggest that the
canine CYP2D15 is polymorphic and phenotyping is needed
in order to assess whether these polymorphisms may play
a critical role in drug metabolism.
quine in human liver microsomes. Among the cDNA-expressed CYPs examined, CYP1A2, 2C8, 2C19, and 2D6
exhibited desethylchloroquine formation with CYP2D6 being the most catalytically competent. The present study demonstrates that CYPs are involved in the metabolism of
chloroquine in human and CYP2D6 is the principal enzyme responsible for chloroquine N-dealkylation in human
liver microsomes.
REFERENCES
1. Sowunmi A., Oduola A.M.: Trans. R. Soc. Trop. Med.
Hyg. 91, 63 (1997).
2. Ducharme J., Farinotti R.: Clin Pharmacok, 31, 257
(1996)
REFERENCES
1. Anzenbacher P., Anzenbacherova E. Cell. Mol. Life
Sci. 58, 737 (2001).
2. Paulson S.K., Engel L., Reotz B., Bolten S., Burton
E.G., Masiasz T.J., Yan B., Schoenhand G.L. Drug Metab. Dispos. 27, 1133 (1999).
3. Sakamoto K, Kirita S, Baba T, Nakamura Y, Yamazoe
Y, Kato R, Takanaka A, Matsubara T. Arch Biochem
Biophys 319, 372 (1995).
WP60 THE INVOLVEMENT OF
CYTOCHROME P450 IN THE
METABOLISM OF
CHLOROQUINE ON HUMAN
LIVER MICROSOMES
WP61 INHIBITION OF CYTOCHROME
P450 ACTIVITIES BY
OLEANOLIC ACID AND
URSOLIC ACID IN HUMAN
LIVER MICROSOMES
KYOUNG-AH KIM, JI-SUK LEE, HI-JOON PARK,
CHANG-JU KIM, IN-SOP SHIM, SEUNG-MOO HAN,
JIN-WOO KIM, and SABINA LIM
Research Group of Pain and Neuroscience, East-West
Medical Research Institute, Kyung Hee University, Seoul,
Korea, [email protected]
KYOUNG-AH KIMa, JI-YOUNG PARKb, JI-SUK LEEa,
and SABINA LIMa
a
Research Group of Pain and Neuroscience, East-West Medical Research Institute, Kyung Hee University, bDepartment of Pharmacology, Gachon Medical School, aSeoul,
bIncheon, Korea, [email protected]
Chloroquine is the first line antimalarial drug for the
treatment and prophylaxis of Plasmodium falciparum infection1. Although chloroquine is thought to be metabolized via the cytochrome P450 (CYP) enzymes2, no data are
currently available on the metabolism of chloroquine in human. Here we evaluated the involvement of CYPs in the
metabolism of chloroquine using human liver microsomes
and cDNA-expressed CYPs. Chloroquine was primarily
metabolized to desethylchloroquine via N-dealkylation
with the apparent Km and Vmax values of 1.03 mM and
0.425 nmol/min/mg protein. Quinidine (a CYP2D6 inhibitor), quercetin (a CYP2C8 inhibitor), and omeprazole
(a CYP2C19 inhibitor) inhibited N-dealkylation of chloro-
S192
Oleanolic acid (OA) and ursolic acid (UA) are triterpenoid compounds that exist widely in food, medicinal
herbs including Achyranthes bidentata1. These substances
have been well studied for the pharmacological activities
including anti-inflammatory, anti-arthritic, and anticancer
activities related to cytochrome P450 (CYP) (i.e. CYP1A2
and CYP2E1) as potential therapeutics2. In our previous
report, extracts of Achyranthes bidentata showed inhibitory effects on CYPs3. So we evaluated whether active
components, OA and UA, are related to the inhibitory effects of Achyranthes bidentata on CYPs using human liver
microsomes. OA competitively inhibited CYP1A2-catalyzed phenacetin O-deethylation and CYP3A4-catalyzed
midazolam 1-hydroxylation with the apparent IC50 (Ki) values of 143.5 (74.2) µM and 78.9 (41.0) µM), respectively.
UA competitively inhibited CYP2C19-catalyzed S-mephenytoin 4’-hydroxylation with the apparent IC50 (Ki) values of 119.7 (80.3) µM. The present study demonstrates
that both OA and UA have inhibitory activities on CYP
isoforms in human liver microsomes and especially OA
may modulate pharmacological and toxicological effects
mediated by CYP1A2 in part. In addition, inhibitory effects of Achyranthes bidentata on CYPs may be related to
that of OA.
This work was supported by Korea Research Foundation Grant and Kyung Hee University. (KRF-2001-005F00024)
ned into pET26b(+) without any tag and expression studies
have just begun. Hopefully, we will be able to obtain all
components of the P450mor system in an active enzymatic
form.
REFERENCES
REFERENCES
1. Jie L, Guo W.J.: World J Gastroenterol 15.493 (2002)
2. Jeong H.G.: Toxicol Lett 105, 215 (1999)
3. Kim K.A., Lee J.S.: Kor J Orient Med (in press)
(2003)
1. Schräder T. et al.: Microbiology 146, 1091 (2000).
2. Sielaff B. et al.: Appl. Microbiol. Biotech. 56, 458
(2001).
3. Poupin P. et al.: Appl. Environ. Microbiol. 64, 159
(1998).
WP62 CLONING, SEQUENCING AND
HETEROLOGOUS EXPRESSION
OF THE GENES CODING FOR
THE P450MOR SYSTEM FROM
MYCOBACTERIUM SP. STRAIN
HE5
WP63 COUMARIN-7-HYDROXYLASE
ACTIVITY IN LIVER AND BRAIN
MICROSOMES OF MICE
BERNHARD SIELAFF, and
JAN REMMER ANDREESEN
Ankara University, Faculty of Pharmacy, Department of
Toxicology, 061 00 Ankara, Turkey
Institute of Microbiology, Martin-Luther-University Halle,
Kurt-Mothes-Str. 3, 06120 Halle, Germany
[email protected]
Coumarin is hydroxylated by the P450 isoform
CYP2A5 in mice liver. CYP2A5 involves in the metabolism of several endogenous and exogenous compounds
such as drugs, carcinogens and environmental pollutants.
Although CYP2A5 is expressed in several tissues, no information is available with respect to brain. Therefore, we
investigated coumarin 7-hydroxylase is present in brain
and compared to that og liver in mice. Accordingly, optimum conditions have been established for both tissues. It
was found that different reaction conditions are required to
achieve maximum enzyme activity in brain and liver of
mice.
OZDAMAR ED., ISCAN M., and EICE BC.
A cytochrome P450 and a ferredoxin, which are specifically induced during growth of Mycobacterium HE5 on
morpholine, piperidine and pyrrolidine, have been purified
(1,2). It was supposed that these proteins are components
of a P450 system responsible for the hydroxylation of these
heterocycles resulting in their ring-cleavage2,3.
The corresponding genes of these proteins were cloned
and sequenced. We identified a gene cluster exhibiting the
genes for P450mor (morC), for the ferredoxin (morF), and
for a putative ferredoxin reductase (morFR), which is
thought to be the third component of this P450 system.
These genes are nearly identical to the genes morA, morB
and morC from Mycobacterium RP1, which were annotated in gene bank recently. This P450 system is also supposed to be involved in the breakdown of the above mentioned N-heterocycles3.
In order to characterize the P450mor system biochemically and to reconstitute an enzymatically active system,
we started to express all three components in E. coli. MorC
was expressed as a fusion protein with an N-terminal or an
C-terminal His-tag. The yields were 190 and 150 nmol
P450/l culture, respectively, and induction was optimal on
addition of 0.75 mM ALA. Unfortunately, we were not
able to record any spectral changes with the purified proteins after addition of the supposed substrates. MorFR was
also cloned in pET28b(+) to obtain an N-terminal His-tag
fusion protein, but we could not detect any expression in
BL21 codon plus. As it seems the protein might be toxic
we will now change to another host strain. MorF was clo-
S193
WP64 CYP4F8 AND CYP4F12:
TISSUE DISTRIBUTION AND
CATALYTIC PROPERTIES
KATARINA STARK, LYDIA SCHAUER,
JOHAN BYLUND, and ERNST H. OLIW
Department of Pharmaceutical Biosciences, Uppsala University, Uppsala University, SE-751 24 Uppsala, Sweden,
[email protected]
Our aims were to compare the tissue distribution and
catalytic properties of CYP4F8 (prostaglandin H 19-hydroxylase) and CYP4F12 in extra hepatic tissues. CYP4F8
was originally cloned from human seminal vesicles and catalyzes 19-hydroxylation of prostaglandin H1 and H2 in
vitro. Antipeptide polyclonal antibodies were raised against the C-termini of the enzymes. Screening of 50 human
tissues for CYP4F8 immunoreactivity revealed protein expression, inter alia, in seminal vesicles, epidermis, corneal
epithelium, sweat glands, the proximal renal tubules, and
epithelial linings of the gut and urinary tract. The CYP4F8
transcripts were detected by Northern blot analysis and RTPCR. A prominent induction of CYP4F8 immunoreactivity
and mRNA was detected in psoriasis in comparison with
unaffected epidermis. CYP4F8 and CYP4F12 occur along
with cyclooxygenases in epithelia, and the transitional
epithelium forms prostacyclin, thromboxane A2 and prostaglandins. Recombinant CYP4F8 metabolised prostacyclin and carbaprostacyclin to their 19-hydroxy metabolites,
which were identified by LC- and GC-MS analysis.
Recombinant CYP4F12 did not metabolise prostacyclin. CYP4F12 immunoreactivity occurred in the epithelial
cells of the gastrointestinal tract (stomach, small intestine,
colon), endothelium of microvessels of placental villi, epidermis, and in the transitional epithelium. Western blot
analysis also suggested that CYP4F12 was expressed in
prostate and purified prostasomes, organelles of prostatic
origin. CYP4F12 mRNA could be detected in placenta,
epidermis, and prostate by RT-PCR.
REFERENCES:
1. Bylund J., Hidestrand M., Ingelman-Sundberg M., Oliw
E.H.: Biol. Chem. 275, 21844 (2000).
2. Oliw E.H., Stark K., Bylund J.: Biochem. Pharmacol.
62, 407 (2001).
3. Stark K., Törmä H., Cristea M., Oliw E.H.: Arch Biochem Biophys. 409(1), 188 (2003).
4. Stark K., Schauer L., Sahlén G., Ronquist G., Oliw
E.H., submitted (2003).
W65
BABOON CYP11B1:
THE EXPRESSION AND
STRUCTURAL ANALYSIS
OF TWO CATALYTICALLY
ACTIVE ISOFORMS
AMANDA C. SWARTa, SANDRA GRAHAMb,
NATASJA BROWNa, and PIETER SWARTa.
a
Dept. of Biochemistry, University of Stellenbosch, Stellenbosch, 7600 South Africa, [email protected], bDept. of
Biochemistry, U.T. Southwestern Medical Center, Dallas,
Texas, 75235
In the adrenal gland the cytochromes P450 catalyse the
synthesis of mineralocorticoids (aldosterone), glucocorticoids (corticosterone and cortisol) and, in some species,
androgens. In human adrenals CYP11B2 catalyses the consecutive 11β-hydroxylation, 18-hydroxylation and 18-oxidation of 11-deoxycorticosterone to aldosterone in the zona
S194
glomerulosa. In the zona fasciculata/reticularis CYP11B1
catalyses the formation of cortisol from 11-deoxycortisol.
CYP11B1 also catalyses the hydroxylation of 11-deoxycorticosterone yielding corticosterone. The subsequent
hydroxylation to 18-hydroxycorticosterone occurs, but to
a lesser degree1. In some species both CYP11B isoforms
are present while in other species a single enzyme catalyses the formation of both glucocorticoids and mineralocorticoids2.
Both CYP11B1 and CYP11B2 occur in the Cape baboon. We have previously reported the existence of three genes encoding baboon CYP11B1 which were cloned from adrenal tissue using RT-PCR. Expression of the recombinant
cDNAs in COS1 cells, co-transfected with the gene encoding human adrenodoxin, showed that two of the cloned genes encoded functional enzymes, while the third was inactive. COS1 cells transfected with the functional cDNAs converted 11-deoxycorticosterone to corticosterone. Negligible
amounts of 18-hydroxycorticosterone and aldosterone were
detected. The conversion of corticosterone to aldosterone
was not observed in the same expression system. We have
also previously shown that deoxycorticosterone was converted to corticosterone in baboon adrenal cortex homogenates.
The metabolism of corticosterone in those preparations was
negligible and aldosterone was not detected3,4.
The three baboon genes share 99% homology and showed 96% and 93% homology with human CYP11B1 and
11B2 respectively. Expression studies in COS1 cells were
subsequently carried out to compare deoxycorticosterone
and deoxycortisol as substrates for the two baboon
CYP11B1 enzymes. In addition, the structures of both
CYP11B1 isoforms were modeled using InsightII (Accelyrs,
Inc). The P450 domain of CYYP102A1 (BM3) and
CYP2C5 were used as the templates for threading the backbone followed by molecular simulation and docking of the
substrate molecule to determine the residues involved in
substrate binding and to correlate them with the two variants
Our results show that the expressed enzymes metabolised the conversion of deoxycorticosterone to corticosterone and deoxycortisol to cortisol respectively, at different
rates when the recombinant baboon cDNAs (babc11a and
babc11) were expressed in COS1 cells in the presence of
recombinant human ADX. The expressed babc11 converted deoxycorticosterone to corticosterone at twice the rate
of the expressed babc11a. Deoxycortisol, however, is converted to cortisol by the two expressed isozymes at a similar rate. The functional genes differ by 3 amino acid residues and structural analyses of the two isozymes may provide valuable information with regards to functional relevance of particular amino acids in the metabolism of specific substrates during the synthesis of mineralocorticoids
and glucocorticoids in the baboon adrenal.
REFERENCES
1. Erdmann B., Gerst H., Bulow H.E., Lenz D., Bahr V.,
Bernhardt R.: Histochem. Cell Biol. 104, 301 (1995).
2. Ohnishi T., Wada A., Lauber M., Yamano T., Okamoto
M.: J. Steroid Biochem. 31, 73 (1988).
3. Swart A.C., Brown N., Kolar N.W., Swart P.: Endocrine
Res. 26, 1011 (2000).
4. Brown N., Swart P., Fenhalls G., Stevens L., Kolar
N.W., Swart A.C.: Endocrine Res. 28, 477 (2002).
WP66 AN INTERSPECIES COMPARISON OF THE METABOLISM
OF THE ANTICANCER
AGENT YONDELIS™
(TRABECTEDIN, ET-743)
MARC VERMEIRa, JAN HENDRICKXa,
ROB HURKMANSa, JOS VAN HOUDTa,
CHRISTINE ZWIJSENa, NINI BODEa, PABLO AVILESb,
IGNACIO MANZANARESb, GEERT MANNENSa,
WILLEM MEULDERMANSa, and
ROLAND DE COSTERa
a
Johnson&Johnson Pharmaceutical Research and Development, a division of Janssen Pharmaceutica N.V., Turnhoutseweg 30, B-2340 Beerse, Belgium, bPharmaMar, Poligono
Industrial La Mina, Avenida de los Reyes 1, E-28770 Colmenar Viejo, Madrid, Spain. [email protected]
Yondelis™ (trabectedin, ET-743) is a potent anticancer
agent isolated from the Caribbean tunicate Ecteinascidia
turbinata1, currently in development for the treatment of
solid tumors2,3. The compound covalently binds in the minor groove of DNA through the 2-amino group of guanine,
bending the DNA towards the major groove4.
The in vitro metabolism of 14C-labelled ET-743 was
studied in liver subcellular fractions of man, male beagle
dog, male Cynomolgus monkey, female New Zealand
white rabbit, male and female Swiss albino mice, and male
and female Sprague-Dawley rats, in the presence of an
NADPH-generating system.
Various metabolites were detected in all the species. The
metabolism of ET-743 in man resembled most that in monkey and was clearly different from that in dog and female
rat. A major yet not fully identified metabolite, designated
metabolite 16, was detected in 12,000 g supernatant fractions of human and monkey. Four metabolites could be identified by both LC-MSMS analysis and co-chromatography
with reference substances. N-Demethylation, with formation
of ET-729, was a major metabolic pathway in the male dog,
the female rat and the female rabbit, and could also be detected in the other species as a minor pathway. Oxidation of
the alcohol function to the carbonyl metabolite ET-759A
was another metabolic pathway that was detected in each
species. Sulphoxide formation (formation of ET-759B) was
detected to a small extent in female mouse only, and hydrolysis of the acetate ester (formation of ET-701) was detected
Fig. 1.
S195
to a small extent in man. The unidentified metabolites in
man were all detected in at least one animal species. These
in vitro metabolism data suggest that the monkey is likely to
represent the most relevant species to evaluate the toxicity of
ET-743, at least from a metabolism point of view.
REFERENCES
Results and discussion
Five hydroxylated metabolites were identified from rat
brain by comigration time and comparison of their absorption
Tab. 1: In vitro metabolites of testosterone by brain microsomes,
in rat and in rabbit. Mean values ± standard deviation.
Metabolites
1. Rinehart K. L., Holt T. G., Fregeau N. L., Stroh J. G.,
Keifer P. A., Sun F., Li L. H., Martin D. G.: J. Org.
Chem. 55, 4512 (1990).
2. Taamma A., Misset J.L., Riofrio M., Guzman C., Brain E.,
Lopez Lazaro L., Rosing H., Jimeno J.M. Cvitkovic E.:
J. Clin. Oncol. 19, 1256 (2001).
3. Villalona-Calero M.A., Eckhardt S.G., Weiss G., Hidalgo M., Beijnen J.H., van Kesteren C., Rosing H.,
Campbell E., Kraynak M., Lopez-Lazaro L., Guzman C.,
Von Hoff D.D., Jimeno J., Rowinsky E.K.: Clin. Cancer
Res. 8, 75 (2002).
4. Zewail-Foote M., Hurley L.H.: J. Med. Chem. 42, 2493
(1999).
WP67 DIFFERENCES IN BIOTRANSFORMATION OF TESTOSTERONE BY BRAIN MICROSOMES
OF RAT AND RABBIT:
A PRELIMINARY STUDY
LUIGI MARVASI, PAOLA ANFOSSI, and
ANNA ZAGHINI
Dipartimento di Sanità Pubblica Veterinaria e Patologia
Animale, Università di Bologna
[email protected]
Rat
Rabbit
6αOH-TST
0.80 ± 0.031
0.32 ± 0.023
6βOH-TST
0.24 ± 0.029
0.18 ± 0.021
16αOH-TST
n.d.
0.07 ± 0.002
16βOH-TST
0.13 ± 0.005
0.15 ± 0.003
11αOH-TST
0.24 ± 0.003
n.d.
2βOH-TST
0.10 ± 0.013
0.03 ± 0.001
n.d: non detectable.
spectra with authentic standards as 6αOH-TST, 6βOH-TST,
11αOH-TST, 16βOH-TST, 2βOH. In rabbit we observed also
the 16αOH-TST but not the 11αOH-TST (Table 1). These
data confirm our previous observations3 according with published results by Rosenbrock4 in rat where 6αOH-TST was
the main metabolite produced by brain microsomes. This
hydroxylated compound was the most representative metabolite also in rabbit even if at lesser concentrations than in
the rat. The biotransformation pattern of TST differs for
brain in relation to liver that produces 6β-hydroxytestosterone as the main metabolite in rabbit5 and in rat6. Although
only few informations are available on P450 isoforms involved in its production in liver and nothing at all in brain,
CYP2A16 and CYP3A187 seem to be involved in the production of 6(OH-TST. Further research could better explain which kind of izoenzimes are involved in this important testosterone biotransformation in the brain.
REFERENCES
Introduction
Even if the liver is the main protagonist in metabolism,
investigation of the extraepatic presence and function of
cytocrome P450 isoenzymes has become a very important
area of interest in recent years. Brain cytochrome P450
ezyzmes are involved in the metabolism of neurosteroids
as well as other, biologically significant, endogenous compounds1. The aim of this study was the investigation on the
main differences in P450 brain activities in two helpful laboratory species, rat and rabbit, using testosterone (TST)
as marker substrate.
Materials and Methods
Brain microsomes were prepared from six males rats
and four male rabbits as described by Voirol 2. TST hydroxylated derivatives (OH-TST) were determined by
HPLC. This study was carried out according to the
Italian Legislation (D.L. 27/01/1992 n° 116) and to
ISO 9001:2000.
S196
Strobel H. W. et al.: Curr Drug Metab. 2, 199 (2001)
Voirol P. et al.: Brain Res, 855, 235 (2000)
Marvasi et al.: Proc. INVITOX Gaeta 2002
Rosenbrock et al.: J Neuroendocrinol, Vol 11, 597
(1999)
5. Zaghini A. et al.: Proc. EAVPT Jerusalem (2000)
6 Sonderfan J. et al.: Arch Bioch and Bioph, Vol 255,
27 (1987)
7 Nagata et al.: J Pharmacog, 6, 103 (1996)
1.
2.
3.
4.
WP68 POLYMORPHISM IN
CYTOCHROME P450
2D6-MEDIATED ACTIVATION OF
MALATHION IN RATS
2. Ahmed R.S., Seth V., Pasha S.T., Banerjee B.D.: Food
Chem. Toxicol. 38, 443 (2000).
3. Sams C., Mason H.J., Rawbone R..: Toxicol. Lett. 116,
217 (2000).
4. Muralidharan G., Hawes E.M., Mc Kay G., Midha
K.K.: Xenobiotica 21, 1141 (1991).
V. SILVARI.a, S. CATANIAa, A. PALAMARa,
L. MARTINO BARBAROb, K. ZAHLSENc, and C. COSTAb
a
Facoltà di Farmacia, Dip. Farmaco-Biologico, Università di
Messina-v.le SS. Annunziata-Messina, Italy, [email protected],
b
Dip. Medicina Sociale del Territorio, Sez. Medicina del Lavoro, Università di Messina-via Consolare Valeria, 1-Messina, Italy, cSt. Olavs University Hospital, Dept. of Clinical
Pharmacology, Trondheim, Norway
Organophosphorus insecticides are widely used in agriculture although they are potentially toxic to humans for
their ability to inhibit the enzyme acetylcholinesterase,
which hydrolyzes the neurotransmitter acetylcholine at
nerve synapse and neuro-muscular junction. They are used
as phosphorothioate, to be converted in vivo to the active
oxygenated form; this process of desulphuration is mediated by cytochrome P450 enzymes1. In the present study we
evaluated the potential involvement of CYP2D6 in malathion biotransformation to the active form malaoxon, which
even seems to induce free radical generation and lipid peroxidation2. CYP2D6 polymorphism may be important in understanding why certain individuals (extensive metabolizers) appear to be at greater risk from exposure to some
organophosphorus compounds than others (poor metabolizers, 5-10 % of Caucasians)3. We used quinine (12.5 mg/kg,
p.o. for 3 days) as a specific inhibitor of CYP2D6 in male
Wistar rats4. The influence on malathion activation was verified in vivo evaluating the inhibition of serum acetylcholinesterase. We also investigated the effects of malathion exposure (1g/kg p.o.) on oxidative stress by assessing lipid peroxidation as malondialdehyde production (MDA), reduced
glutathione (GSH) consumption, glutathione-S-transferase
(GST) enzymatic activity, variation of catalase (CAT) and
superoxide dismutase (SOD) in liver homogenate of rats
treated with quinine and malathion. Acetylcholinesterase
inhibition and lipid peroxidation determined by malathion
were significantly reduced by quinine treatment, which also
reduced GSH consumption and GST, CAT and SOD increment due to malathion. In conclusion, these data confirm
that oxidative stress is involved in the toxicity of malathion
and support the hypothesis that CYP2D6 has an important
role in the bioactivation pathway of this pesticide. As a consequence, the polymorphism of CYP2D6 influences individual susceptibility to exposure to malathion.
REFERENCES
1. Gallo M.A., Lawrik N.J. in: Handbook of Pesticide Toxicology (Hayes W.J., Laws E.R., ed.), vol. 2, pp. 9171123. Academic, Press, San Diego 1991.
S197
WP69 IN VITRO STUDIES ON THE
ACTIVATION OF THE
ORGANOPHOSPHATE
PESTICIDE MALATHION BY
CYTOCHROME P450 2D6 IN RAT
LIVER MICROSOMES
C. COSTAb, S. CATANIAa, V. D’ANGELOa,
L. MARTINO BARBAROb, K. ZAHLSENc, and
V. SILVARIa
a
Facoltà di Farmacia, Dip. Farmaco-Biologico, Università di
Messina-v.le SS. Annunziata-Messina, Italy, [email protected],
b
Dip. Medicina Sociale del Territorio, Sez. Medicina del Lavoro, Università di Messina-via Consolare Valeria, 1-Messina, Italy, cSt. Olavs University Hospital, Dept. of Clinical
Pharmacology, Trondheim, Norway
Malathion is one of the phosphorothioate pesticides
mostly used in agriculture and public health programmes.
It has been detected in the environment as contaminant of
drinking water and pesticide residues foodstuff; thus, human exposure to malathion may occur through occupational settings, agricultural workers or pesticide applicators,
as well as via a variety of environmental sources.
Malathion is a weak acetylcholinesterase inhibitor unlike its metabolite, malaoxon, which is a potent anticholinesterase agent. Moreover, recent studies raised concerns
over the potential of malathion and its metabolite to cause
genetic damage as strongly positive alkylating agents. As
well known the activation of malathion to the oxygen analogue malaoxon occurs in vivo by the oxidative desulfuration of the parent compound and it is mediated by the cytochromes P450 (CYP), even if the specific isoform involved in the metabolizing process is not yet known.
In the present in vitro study we used quinine, a specific
inhibitor of CYP2D6, to investigate if malathion activation
in rat liver microsomes is catalysed by CYP2D6 subfamily.
If this hypothesis is confirmed proving that CYP2D6 is involved in malathion activation, its inhibition by quinine
may show a reduction of the oxon genotoxicity on human
leucocytes in the single cell gel electrophoresis (comet assay), which is a sensitive genotoxicity test able to investigate DNA damage detecting single strand breakage. In the
present study the results show that acetylcholinesterase inhibition and genotoxicity determined by malathion were
significantly reduced by quinine treatment, due to a decre-
ment of malaoxon production, suggesting that CYP2D6
may have an important role in the bioactivation pathway of
this pesticide.
According to the knowledge that CYP2D6 is characterized by a genetic polymorphism, it follows that if
CYP2D6 is responsible of malathion activation the risk deriving from exposure to malathion may be dependent on
the genotype of the population. As a consequence, the polymorphism of CYP2D6 influences individual susceptibility to exposure to malathion.
REFERENCES
1. Titenko-Holland N., Windham G., Kolachana P., Reinisch F., Parvatham S., Osorio A.M., Smith M.T.: Mutat. Res. 15, 388, 85 (1997).
2. Blasiak J., Jaloszynski P., Trzeciak A., Szyfter K.: Mutat. Res. 30, 445, 275 (1999).
3. Muralidharan G., Hawes E.M., Mc Kay G., Midha
K.K.: Xenobiotica 21 (11), 1141 (1991).
4. Sams C., Mason H.J., Rawbone R..: Toxicol. Lett. 116,
217 (2000).
WP70 PHOTOAFFINITY LABELING OF
CYTOCHROME P450 2B4
MARTIN KARABEC, MIROSLAV ·ULC,
and PETR HODEK
University, Albertov 2030, 128 40 Prague 2, The Czech Republic, [email protected]
Photoaffinity labeling is a chemical modification technique used to study protein structure and interactions.
Highly reactive intermediate which is able to modify
amino acid residues of a protein is generated by UV-light
irradiation at certain place and exact time. The goal of this
technique is the identification of protein active centre or
binding sites. Diamantane like compounds are highly specific substrates of phenobarbital-inducible cytochrome
P450 2B4. Their affinity for the active centre is documented by spectral dissociation constants: adamantane,
Ks=1.3x10-6 mol/l; diamantane, Ks=0.5x10-6 mol/l; triamantane, Ks=4.3x10-6 mol/l. Diamantane has the highest affinity for an active centre of cytochrome P450 2B4. Hence,
diamantane skeleton was used for the construction of photoaffinity label. Its photolabile derivative, 3-azidiamantane
[spiro-(diazirine-3,3’-diamantane)] was synthetized and
evaluated.
3-Azidiamantane
dissociation
constant
Ks=1.9x10-6 mol/l proves its affinity is comparable to diamantane. It guarantees effective binding to the active
centre and labeling. Two UV-light sources for the label
photoactivation were compared, first emitting light of
254nm and second 366nm. Photo activation of 90% of 3-
S198
azidiamantane by the 254nm source is accompanied by destroying more then 20% of cytochrome P450 2B4, while by
366nm one less then 10%. Upon photolysis (366nm, half
live 1.5 minute) 3-azidiamantane gives highly reactive carbene intermediate. Photolysis of 3-azidiamantane in hexan
results in formation of 3-hexyldiamantane (48.3%), demonstrating excellent incorporation of carbene intermediate to unactivated C-H bounds. When photoaffinity label
was photolysed in water, the major products were 3-hydroxydiamantane (82.0%, Ks=49.2x10-6 mol/l) and 3-diamantanone (14.3%, Ks=4.6x10-6 mol/l); those side products of
an active intermediate might interfere with active centre labeling. The experiment for photoaffinity labeling is conducted in three arrangements: photolysis of cytochrome
P450 2B4, cytochrome P450 2B4 with 3-azidiamantane
and cytochrome P450 2B4 in the presence both 3-azidiamantane and diamantane, used as a competitor to differentiate specifically bounded label in active center and label
bound outside. Labeled cytochrome P450 2B4 is cleaved
by trypsine and peptides are separated on HPLC RP C18.
Separated peptides from all three arrangements are analyzed on MALDI TOF and MS-DECA and results are compared to distinguish peptides labeled by diamantane originating from the cytochrome P450 2B4 active centre, from
those labeled nonspecifically.
This study was supported by Grant MSM-113100001
from Ministry of Education of the Czech Republic.
8
Tab. 1.: Examples of Constitutional DME Allele Frequencies of
Non Cancer Patients and Breast Cancer Patients
LATE SUBMISSION – POSTER
GENOTYPING OF DRUG
METABOLIZING ENZYMES IN
DNA OF ARCHIVED TISSUES
FOR FUTURE ASESSMENT OF
BREAST CANCER DRUG
TREATMENT RESPONSE AND
NON RESPONSE
MALGORZATA JAREMKO, PETER FRITZ,
CHRISTINA JUSTENHOVEN, and
HILTRUD BRAUCH
Dr. Margarete Fischer - Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany, [email protected]
According to current scientific concepts polymorphisms
of drug metabolizing enzymes and other potential drug targets may play a role in inter-individual differences in treatment responses. This is of major concern in breast cancer,
a disease that kills more than 300,000 women each year
worldwide. Constitutional variations in drug response, non
response and toxicity may be studied on the genome level by
comparing allele/genotype frequencies of potentially relevant drug metabolizing enzymes (DME) between breast cancer patient groups, i.e. treatment responders and non responders. In addition, these studies require long-term clinical follow-up under hormonal treatment, chemo- and immunotherapy, respectively. However, although clinical and survival
data may be available, there is frequently no blood DNA
available from eligible patients for genotyping. Therefore,
these studies must rely on the genotyping of DNA isolated
from formalin fixed and paraffin embedded regular breast
tissues. At present it is not clear, if these tissues will be suitable for genotyping analyses since there has been frequently
observed failure in PCR-based analyses of archival DNA
due to poor quality. Here we tested the applicability of Taqman technology based allelic discrimination for the genotyping of DME, i.e. cytochrome P450: CYP2C19 (*2)1,2,
CYP2D6 (*3, *4,*6, *7, *8)3,4,5,6,7, CYP2C9 (*2,*3)8 and
EPHX9 involved in the metabolism of breast cancer drug treatments. We included paraffin embedded normal tissues
of 44 non-cancer patients collected from 1992-2002, and
74 breast cancer patients collected from 1986-1988 (fiveyear survival: 18 patients without metastasis, 4 patients with
metastasis, and 27 deceased). DNA was isolated from 6 to
30 10 µm tissue sections with the QIAamp(r) DNA Mini Kit
and DNA amounts obtained ranged from 18 to 334 µg/ml.
All allele/genotype frequencies tested so far could be called
in all DNA samples and genotype frequencies of non-cancer
patients were in Hardy-Weinberg equilibrium and comparable to published frequencies 1,2,4,5,6,9. Results of allele
frequencies of CYP2D6 and CYP2C19 are listed in table 1.
We observed a significantly higher frequency of the mutant
S199
CYP2D6*3 allele (delA) in breast cancer patients when
compared to non-cancer patients (OR 8.28; CI 1.043-65.758;
P=0.0287). This may indicate that breast cancer patients
may be more frequently compromised in CYP2D6 enzymatic function. Our data suggest that DNA from formalin fixed
and paraffin embedded tissues is suitable for Taqman technology based allelic discrimination analysis. Differences in
allele/genotype frequencies may be observed when comparing patient groups. It will now be possible to apply this
technology towards the genotyping of CYP2C19, CYP2D6,
CYP2C9 and EPHX in large archived breast cancer tissue
collections for future studies on inter-individual differences
in breast cancer treatment response.
REFERENCES
1. Roddam P.L., Rollinson S., Kane E., Roman E., Moorman A., Cartwright R., Morgan G.J.: Pharmacogenetics
10, 605 (2000).
2. Wedlund P.J.: Pharmacology. 61, 174 (2000).
3. Buchert E.T., Woosley R.L., Swain S.M., Oliver S.J.,
Coughlin S.S., Pickle L., Trock B., Riegel A.T.: Pharmacogenetics. 3, 322 (1993).
4. Broly F., Marez D., Lo Guidice J.M., Sabbagh N., Legrand
M., Boone P., Meyer U.A.: Hum.Genet. 96, 601 (1995).
5. Leathart J.B., London S.J., Steward A., Adams J.D.,
Idle J.R., Daly A.K.: Pharmacogenetics. 8, 529 (1998).
6. Saxena R., Shaw G.L., Relling M.V., Frame J.N., Moir
D.T., Evans W.E., Caproso N., Weiffenbach B.: Human.
Mol. Genet. 3, 923 (1994).
7. Zanger U.M., Fischer J., Raimundo S., Stuven T., Evert
B.O., Schwab M., Eichelbaum M.: Pharmacogenetics.
11, 573 (2001).
8. Stubbins M.J., Harries L.W., Smith G., Tarbit M.H.,
Wolf C.R.: Pharmacogenetics. 6, 429 (1996).
9. Cortessis V., Siegmund K., Chen Q., Zhou N., Diep A.,
Frankl H., Lee E., Zhu Q.S., Haile R., Levy D.: Cancer
Research. 61, 2381 (2001).
06 ForumYS.QXD 10.6.2003 11:55 Stránka 200
Forum of Young Scientists – FYS
9
FORUM OF YOUNG SCIENTISTS – FYS
FYS02 LUMINESCENT MOLECULAR
PROBES FOR CYTOCHROME
P450
FYS01 HIJACKING E. COLI’S
FERRICHELETASE: EFFICIENT,
BIOSYNTHETIC AND
NON-DENATURATING
PREPARATION OF COBALT
P-450 ENZYMES
ALEXANDER R. DUNNa,
ANNA-MARIA A. HAYSb, DAVID B. GOODIb,
C. DAVID STOUTb, RICHARD CHIUa,
JAY R. WINKLERa, and HARRY B. GRAYa
a
Beckman Institute, California Institute of Technology, Pasadena, California 91125, USA, and bDepartment of Molecular Biology, The Scripps Research Institute, 10550 North
Torrey Pines Road, La Jolla, California 92037, USA,
[email protected]
WESLEY E. STRAUB, and
PAUL R. ORTIZ DE MONTELLANO,
Department of Pharmaceutical Chemistry, University of
California, 600 16th Street, San Francisco, CA 941432280, USA, [email protected]
Denaturing methodologies have been used to exchange
the porphyrin of a wide number heme proteins for decades,
but success with P-450s has been limited. Rather than unfolding and re-constituting a wild-type protein with an iron
heme, we report an efficient and biosynthetic method of
preparing cobaltic CYP101 and CYP119. Heterologous expression of the P-450 genes on metal deficient M9 minimal
media underlies the method. Substitution of iron with cobalt causes the porphyrin to be more labile and decreases
enzyme thermal stability. High resolution crystal structures
indicate this perturbation is likely due to an increased cobalt sulfur bond length. In contrast, nitrogen ligands such
as imidazole and 4-phenyl imidazole bind with higher affinity to the cobalt CYP101 and CYP119. Exploiting the diamagnetic status of water bound CYP119, 2D NMR experiments were used to observe multiple conformations of
CYP119 in solution. As revealed by earlier X-ray structures, the binding of imidazoles shifts the equilibrium of
multiple conformers to one conformation which is open or
closed. Iron CYP119 but not cobalt CYP119 hydroxylates
lauric acid using the peroxide shunt. Hydroxylation activity by cobalt CYP119, however, was observed using
NADPH and electron transfer proteins. In the presence of
hydrogen and alkyl peroxides, cobalt CYP101 reacts quantitatively to yield porphyrin adducts which decompose into
stable hydroxy porphyrins, a reactions likely driven by the
unique radical chemistry of cobalt porphyrins. When expanded to a wider scope of P450 enzymes and transition
metals, the biosynthetic preparation of metal substituted
P450 enzymes promises to open new areas of both P450
chemistry and spectroscopy. Work supported by NIH
Grants GM25515.
Reduction of the ferrous-dioxygen intermediate of cytochrome P450cam by putidaredoxin is the rate-determining step in the catalytic cycle. In order to study the fleeting intermediates in P450 catalysis, we have developed
a series of ruthenium-diimine based photoreductants that
bind to the active site of P450cam and reduce the heme
upon 470-nm excitation (Fig. 1a). The variation in electron
transfer (ET) rates among the Ru-diimine:P450 conjugates
strongly supports the through-bond electronic coupling
model for the Ru:heme system. Light-triggered ET allows
temporal resolution of events on the nanosecond timescale,
100 times faster than the timescales achieved by stoppedflow mixing.
Two dansyl-based, luminescent probes (D-4-Ad and
D-8-Ad) were synthesized that target P450cam (Fig. 1b).1
D-4-Ad luminescence is quenched by Förster energy transfer upon binding (Kd = 0.83 µM), but is restored when the
probe is displaced from the active site by camphor. In contrast, D-8-Ad (Kd ~ 0.02 µM) is not displaced from the enzyme even in the presence of a large excess of camphor.
Probes with properties similar to those of D-4-Ad potentially could be useful for screening P450 inhibitors. The re-
Fig. 1. Crystal structures of a Ru-diimine photosensitizer (A) and
D-8-Ad (B) bound to cytochrome P450cam.
S200
markable binding affinity of D-8-Ad suggests novel P450
inhibition strategies.
Crystal structures D-4-Ad, D-8-Ad, and Ru-diimine
complexes2 bound to cytochrome P450cam show that the
probes capture the enzyme in three different conformational states. Conformational flexibility in the B’, F and G helices allow the enzyme to bind molecules much larger than
the natural substrate camphor. Large changes in peripheral
enzyme structure (F and G helices) couple to conformational changes in active center residues (I helix) implicated in
proton pumping and dioxygen activation. Structural flexibility in the F/G helix region likely allows the 54 human
P450s to oxidize thousands of substrates.
REFERENCES
1. Dunn A. R., Hays A.-M. A., Goodin D. B, Stout C. D.,
Chiu R., Winkler J. R., Gray H. B.: J. Am. Chem. Soc.
124, 10254 (2002).
2. Dunn A. R., Dmochowski I. J., Bilwes A. M., Gray H.
B., Crane B. R.: Proc. Natl. Acad. Sci. USA 98, 12420
(2001).
FYS03 AUTOMATED DOCKING OF
LIGANDS TO CYTOCHROME
P450 ENZYMES
first aim was to investigate which automated docking strategies are most appropriate for the refinement and validation of our human P450 homology models.
Using 39 X-ray crystal structures of P450-ligand complexes deposited at the Protein Databank as a test set, we
evaluated several different docking algorithms (AutoDock2
(simulated annealing (SA), ‘Darwinian’ genetic algorithm
(GA) and ‘Lamarckian’ genetic algorithm (LGA)), FlexX3
(incremental construction algorithm) and GOLD4 (‘Darwinian’ genetic algorithm)) in combination with corresponding and additional (SCORE5, X-CSCORE6 and PMF7)
scoring functions, and determined which docking approaches are most reliable in predicting the binding modes and
binding affinities of these systems. In most of the cases studied, the usage of different charge models, incorporation of
covalent binding information, and increasing the number
of docking runs and enlarging the binding cavity compared
to standard values did not have any significant effect on the
performance of different docking approaches. In many cases, including crystal waters also had no or minor influence
on docking accuracy. The GOLD-SCORE and Autodock
(GA)-SCORE docking-scoring combinations seemed to be
the most appropriate with respect to structure prediction,
while no clear correlations were found between predicted
and experimentally determined binding affinities of 16
P450-ligand complexes (neither by scoring of X-ray structures, nor docked poses).
REFERENCES
CHRIS DE GRAAFa, PETER H.J. KEIZERSa,
JAN N.M. COMMANDEURa, SASKIA M. VAN DER
VIESb, and NICO P.E VERMEULENa
a
LACDR, Division of Molecular Toxicology, Department of
Pharmacochemistry, bDepartment of Biochemistry and
Molecular Biology, Vrije Universiteit, De Boelelaan 1083,
1081 HV Amsterdam, the Netherlands
[email protected]
In the absence of experimentally determined structures,
homology models of human P450 isoenzymes CYP1A1,
CYP1A2 and CYP2C9 are constructed using the only available mammalian P450 structure crystal structure (rabbit
CYP 2C5)1 as a template. These computer models are
being validated and refined using automated docking,
MD- simulations and essential dynamics (ED) analysis in
combination with experimental data provided by mutation
studies, SERRS and spin relaxation NMR. Automated docking methods can be used to predict energetically favorable
conformations and orientations (poses) of (potential) ligands in the interior structure (binding cavities) of a protein. These methods consist of both an algorithm to generate different poses (docking), and a scoring function to
subsequently consider the tightness of protein-ligand interactions (e.g., discrimination of favorable poses from unfavorable ones). Several docking algorithms and scoring
functions have been described the past few years, so our
S201
1. Williams P.A., Cosme J., Sridhar V., Johnson E.F.,
McRee D.E.: Mol. Cell 5, 121 (2000)
2. Morris G.M., Goodsell D.S., Halliday R.S., Huey R.,
Hart W.E., Belew R.K., Olson A.J.: J. Comp. Chem. 19,
1639 (1998)
3. Rarey M., Wefing S., Lengauer T.: J. Comp.-Aid. Mol.
Des 10, 41 (1996)
4. Jones G., Willett P., Glen R.C., Leach A.R., Taylor R.:
J. Mol. Biol. 26, 7727 (1997)
5. Wang R.X., Liu L., Lai L.H., Tang Y.Q.: J. Mol. Model.
4, 379 (1998)
6. Wang R.X., Lai L.H., Wang S.M.: J. Comp.-Aid. Mol.
Des. 16, 11 (2002)
7. Muegge I, Martin Y.C.: J. Med. Chem. 42, 791 (1999)
FYS04 CALMODULIN ACTIVATES
INTERFLAVIN ONE-ELECTRON
TRANSFER IN THE HUMAN
NEURONAL NITRIC OXIDE
SYNTHASE FLAVIN DOMAIN
FYS05 ELECTRON TRANSFER
IN FLAVOCYTOCHROME P450
BM3 AND THE C999A MUTANT:
KINETICS OF FLAVIN
REDUCTION AND
RELATIONSHIPS WITH
MAMMALIAN CYTOCHROME
P450 REDUCTASE.
ZHI-WEN GUAN, YOSHITAKA NISHINO,
KEITA YAMAMOTO, SHIGENOBU KIMURA,
and TAKASHI IYANAGI
Hyogo 678-1297 Japan: [email protected]
The flavin semiquinones, FADH. and the FMNH. participate in the regulation of one-electron transfer within the
NOS flavin domain1, 2. The FAD semiquinone form was unstable in the presence of O2, and showed a characteristic
absorption peak at 520 nm, which was not observed in the
FMN semiquinone1. This makes possible to monitor the
proportion of FAD semiquinone generated and directly determine the rate of one-electron transfer between the two
flavins using stopped-flow spectrophotometry. CaM binding results in a greater than 10-fold increase in the rate of
interflavin (i.e. FAD ■ FMN) one-electron transfer.
The reduction of the air-stable semiquinone (FADFMNH.) of both CaM-bound human nNOS and iNOS flavin domains with NADPH showed that the extent of a conversion of FADH2/FMNH. to FADH./ FMNH2 in the iNOS
flavin domain was greater than that of the nNOS flavin domain. The reduction of both oxidized domains (FAD-FMN)
with NADPH resulted in the initial formation of small
amount of disemiquinone, followed by its decay. The rate
of interflavin electron transfer between the two flavins in
the iNOS flavin domain was faster than that of the nNOS
flavin domain. In addition, the formation of a mixture of
the two- and four-electron reduced states in the presence of
excess NADPH was different for the two NOS flavin domains. These data indicate a more favorable formation of
the active intermediate, FMNH2 in the iNOS flavin domain, as compared with the nNOS flavin domain. This may
explain why the iNOS flavin domain has a high activity towards electron acceptors.
REFERENCES
1. Guan,
Z-W.,
Iyanagi,
T.:
Arch.
Biochem.
Biophys. 412, 65 (2003).
2. Matsuda, H., Iyanagi, T.: Biochim. Biophys. Acta 1473,
345 (1999).
S202
OLIVIER ROITEL, NIGEL S. SCRUTTON and
ANDREW W. MUNRO
Department of Biochemistry, University of Leicester, The
Adrian Building, University Road, Leicester LE1 7RH, UK
The cytochromes P450 are a superfamily of haem-containing mono-oxygenase enzymes found in all major domains of life. They catalyse the reductive scission of dioxygen,
and generate an oxygenated product, water and NADP + 1.
The diflavin enzyme cytochrome P450 reductase (CPR)
supplies electrons to eukaryotic P450 enzymes 2. CPR has
arisen by the fusion of ancestral domains related to flavodoxin and NADP+-ferredoxin oxidoreductase3. Flavocytochrome P450 BM3 comprises a prokaryotic CPR and a soluble P4504. The diflavin reductase of flavocytochrome
P450 BM3 is fused to a P450 fatty acid hydroxylase, and
the enzyme has high levels of activity towards a range of
long chain fatty acid substrates5. The crystallographic structure of the haem domain of P450 BM3 has been solved in
the presence and absence of bound substrate 7,8, and solution
studies have clarified the roles of a number of active site residues in substrate binding and catalysise.g. 6,9. Structural and
mechanistic studies of the diflavin reductase domains of flavocytochrome P450 BM3 are less advanced than for the
P450 domain.
The primary structure of P450 BM3 reductase lacks the
membrane anchor found in mammalian enzymes. Despite
the inferred structural similarities, the thermodynamic properties of BM3 reductase are distinct from human and rabbit CPR, suggesting major differences in the catalytic cycles of the bacterial and mammalian enzymes10-12. BM3 reductase catalyses the reduction of artificial electron acceptors (e.g. cytochrome c and ferricyanide) more effectively
than its eukaryotic counterparts. It also reduces its own
heme domain at rates in excess of 200 s-1 13, which far surpasses the reduction rates of mammalian P450s by their
cognate CPRs 14. The ability of BM3 reductase to deliver
electrons rapidly to its P450 domain is due, in part, to the
fast reduction of FAD by NADPH, which is considerably
slower in mammalian CPR 15. Notwithstanding, several residues known to facilitate FAD reduction by NADPH
in mammalian CPRs are conserved in BM3 reductase.
These include the „catalytic triad“ identified by Kasper and
co-workers in rat CPR, comprising Ser 457, Cys 630 and
Asp 675 16. These are equivalent to Ser 830, Cys 999
and Asp 1044 in BM3 reductase17. In the rat C630A mutant
CPR, the cytochrome c reductase rate is decreased approximately 50-fold16 and reduction of the flavin cofactors by
NADPH is impaired. It has been suggested that Cys-630
either destablilizes the nicotinamide C4-H bond and/or donates a proton to the N5 atom of FAD during enzyme reduction16. Also, it has been suggested that flavin reduction
is thermodynamically disfavoured in rat C630A CPR relative to wild-type16.
Alignment of the available sequences for diflavin reductases indicates that Cys-630 of rat CPR is conserved
throughout the family of proteins, except for human NR1.
Modelling of the FAD domain of flavocytochrome P450
BM3 indicates that Cys-999 likely occupies a position analogous to that of Cys-630 in rat CPR. Given the predicted
importance of Cys-999 (by analogy with Cys-630 of rat
CPR), and that flavin reduction in flavocytochrome P450
BM3 is much faster than that seen with other diflavin reductasese.g. 13, 15, we have examined the kinetic and thermodynamic properties of the C999A flavocytochrome P450
BM3. We show that the rate of FAD reduction is substantially impaired in C999A flavocytochrome P450 BM3. We
provide evidence for a second, kinetically distinct nicotinamide coenzyme binding site, the occupation of which attenuates the rate of NADP+ reduction by reduced flavocytochrome P450 BM3. We suggest that NADP+ release from
reduced flavocytochrome P450 BM3 is faster than with
mammalian CPR. Unlike with mammalian CPR, this
accounts for the lack of attenuation of the FAD reduction
rate by NADPH through occupation of the second NADPH
binding site, and this contributes to the superior catalytic
rate of BM3 reductase. These data further our understanding of the complex phenomena underlying electron transfer and its control in the CPR family of enzymes.
REFERENCES
1. Munro A.W., Lindsay J.G.: Mol. Microbiol. 20, 1115
(1996).
2. Iyanagi T., Mason H.S.: Biochemistry 12, 2297 (1973).
3. Wang M., Roberts D.L., Paschke R., Shea T.M., Masters B.S.S., Kim J.-J. P.: Proc. Natl. Acad. Sci. USA 94,
8411 (1997).
4. Narhi L.O., Fulco A.J.: J. Biol. Chem. 262, 6683
(1987).
5. Narhi L.O., Wen L.P., Fulco A.J.: Mol. Cell. Biochem.
79, 63 (1988).
6. Noble M.A., Miles C.S., Chapman S.K., Lysek D.A.,
Mackay A.C., Reid G.A., Hanzlik R.P., Munro A.W:.
Biochem. J. 339, 371 (1999).
7. Ravichandran K.G., Boddupalli S.S., Hasemann C.A.,
Peterson J.A., Deisenhofer J.:. Science 261, 731 (1993).
8. Li H.Y., Poulos T.L.: Nature Struct. Biol. 4, 140 (1997).
9. Ost T.W.B., Miles C.S., Munro A.W., Murdoch J., Reid,
G.A., Chapman S.K.: Biochemistry 40, 13421 (2001).
10.Munro A.W., Noble M.A., Robledo L., Daff S., Chapman, S.K.: Biochemistry 40, 1956 (2001).
S203
11.Iyanagi T., Makino N., Mason H.S.: Biochemistry 13,
1701 (1974).
12.Daff S.N., Chapman S.K., Turner K.L., Holt R.A., Govindaraj S., Poulos T.L., Munro A.W.: Biochemistry 36,
13816 (1997).
13.Munro A.W., Daff S., Coggins J.R., Lindsay J.G.,
Chapman S.K.: Eur. J. Biochem. 239, 403 (1996).
14.Guengerich F.P., Johnson, W.W.: Biochemistry 36,
14741 (1997).
15.Gutierrez A., Lian L.-Y., Wolf C.R., Scrutton N.S., Roberts, G.C.K.: Biochemistry 40, 1964 (2001).
16.Shen A.L., Sem D.S., Kasper, C.B.: J. Biol. Chem. 274,
5391 (1999).
17.Govindaraj S., Poulos, T.L.: J. Biol. Chem. 272, 7915
(1997).
FYS06 DIRECTED EVOLUTION OF
ALKANE HYDROXYLATION
ACTIVITY IN CYTOCHROME
P450 BM-3
PETER MEINHOLDa, MATTHEW W. PETERSa,
EDGARDO T. FARINASa, ANTON GLIEDERb,
SARINA MOHANTYa and FRANCES H. ARNOLDa
a
Division of Chemistry and Chemical Engineering, California Institute of Technology, Mail Code 210-41, Pasadena, CA 91125, USA, [email protected]
b
Institute of Biotechnology, Technical University of Graz,
Petersgasse 12, A-8010 Graz, Austria
Cytochrome P450 BM-3 (CYP102) from Bacillus megaterium is a soluble, self-sufficient, highly active (C12C18) fatty acid monooxygenase. We have recently converted this enzyme into a highly efficient catalyst for the conversion of alkanes to alcohols 1. The mutant enzyme exhibited initial rates faster than any other previously reported
alkane hydroxylase. Using this mutant as a starting point
we have applied directed evolution techniques and site-directed mutagenesis to further improve activity on short, gaseous alkanes such as propane, ethane and methane. Catalyzing this reaction requires efficient substrate binding and
shifting the regioselectivity towards terminal hydroxylation. For this purpose we developed a colorimetric screening assay based on the conversion of alkyl methyl ethers
that allows for detection of increased turnover number as
well changes in regioselectivity. Propane-oxidizing mutant
enzymes have been generated that show increased total turnover number without a shift in regioselectivity. However,
these mutants do exhibit a shift in regioselectivity towards
terminal oxidation in the hydroxylation of octane. The demonstrated ability of the P450 BM-3 framework to accommodate these enhanced activities towards small alkanes
suggests that methane hydroxylation activity might be
accessible to this system upon the application of further
rounds of directed evolution.
REFERENCES
1. Glieder A., Farinas E. T., Arnold, F. H.: Nat. Biotech.
20, 1135 (2002).
FYS08 REGULATION OF EXPRESSION
OF THE HUMAN LANOSTEROL
14α-DEMETHYLASE GENE
(CYP51)
MARTINA FINK, and DAMJANA ROZMAN
Medical Center for Molecular Biology, Institute of Biochemistry, Medical Faculty University of Ljubljana, Slovenia,
[email protected]
FYS07 DIRECTED EVOLUTION
OF P450 BM-3 FOR
BIOTRANSFORMATIONS
TUCK SENG WONG, and ULRICH SCHWANEBERG
International Universoty Bremen (IUB), Bremen, Germany
Cytochrome P450 BM-3 (EC 1.14.14.1) catalyses hydroxylation and epoxidation of a broad range of valuable
compounds including alkanes, alcohols, fatty acids, amides, polyaromatic hydrocarbons and heterocycles, reactions which are difficult to be achieved by chemical means.
However, the Km values of P450 BM-3 towards most of
these notoriously insoluble compounds fall in mM range,
which greatly limits its practical application. An organic
co-solvent is thus necessary to increase the substrate solubility and to achieve a higher catalytic efficiency. Applying
P450 BM-3 in such non-natural working environment requires increase of its tolerance towards organic co-solvents. By directed evolution using pNCA assay1,2, we improved the organic co-solvent resistance of P450 BM-3 towards DMSO and THF with increases in specific activity
of: up to 10-fold in 2 % (v/v) THF and up to 6-fold in
10 % (v/v) DMSO3. The evolved mutants also showed
a significantly higher tolerance towards other organic cosolvents including acetone, acetonitrile, DMF and ethanol3. In addition, colorimetric assays based on 4-aminoantipyrine, Fast Violet B salt and Fast Blue RR salt are being
developed to detect hydroxylated aromatic biotransformation products and to access the industrial potential of organic co-solvent resistant mutants.
REFERENCES
1. Schwaneberg U., Schmidt-Dannert C., Schmitt J.,
Schmid R. D.: Anal. Biochem. 269, 359 (1999).
2. Schwaneberg U., Cirino P. C., Otey C., Farinas E.,
Arnold F. H.: J. Biomol. Screen. 6, 111 (2001).
3. Schwaneberg U., Wong T.S., Arnold, F. H.: (submitted)
(2002)..
S204
Lanosterol 14α-demethylase (CYP51) is the most conserved cytochrome P450 with the major role in cholesterol
biosynthesis in mammals. Having a housekeeping role in
cholesterol biosynthesis, CYP51 it is expressed ubiquitously, with highest levels observed in the testis, liver,
ovary and the adrenal gland. These tissues differ in the
sense whether they mainly produce cholesterol and excrete
it in the form of bile acids (liver), or require cholesterol for
steroid hormone biosynthesis (testis, ovary, adrenal gland).
In addition to steroid hormones, testis and ovaries accumulate intermediates of the cholesterol biosynthetic pathway
that are not observed in the liver. Meiosis activating sterols
(MAS) are short-lived products of CYP51 demethylation
reaction in somatic cells, but accumulate in gonadal tissues, suggesting their role in reproduction. The different
roles of cholesterol and proposed roles of its intermediates
suggest that regulation of enzyme activities of cholesterogenic genes differ between tissues. Consequently, it is expected that transcriptional regulation of genes encoding
cholesterogenic enzymes is mediated in a tissue-specific
manner. The complex promoter/regulatory region of mammalian CYP51 genes permits trans-activation by a variety
of signalling pathways, resulting is a tissue-specific mode
of regulation. Sterol regulatory element (SRE) binds sterol
regulatory element binding proteins (SREBPs) that are
common trans-activatiors of cholesterogenic genes in the
liver and mediate the negative cholesterol feedback signaling. SREBPs bind to SRE elements in low-cholesterol
conditions, resulting in trans-activation of genes, including
CYP51. When cholesterol is not limiting to the cell,
SREBP-dependent genes are expressed only at basal level.
To perform the trans-activation function, SREBPs require
interactions with other transcription factors and with co-regulatory proteins, that different from gene to gene. In the
case of the human CYP51 gene we demonstrated SREBP
interactions with Sp1 and with CRE-binding proteins.
cAMP-responsive element (CRE2) binds cAMP-dependent
transcription factors and is necessary for SREBP-dependent transcriptional activation. SREBP-1a is phosphorylated by PKA in ex vivo conditions and can interact with different co-activator proteins, such as CREB-binding protein
(CBP) and activator of CREM in testis (ACT). Both mentioned co-activator proteins are not expressed in identical
tissues, suggesting that SREBP transcriptional activity is
modulated in different tissues according to choice/availabi-
lity of co-regulatory and coactivator proteins. The cAMPdependent transcriptional activation of CYP51 is from the
physiological standpoint likely more important in germ
cells and in adrenal gland compared to the liver. We have
shown that a cAMP-dependent trans-activation of CYP51
is acieved (by cAMP-dependent stimuli, such as forskolin,
or by overexpression of transcription factors CREB and
CREM) independently of the SREBP-dependent pathway.
CYP51 transcription is activated ex vivo in sterol-repressed
conditions, when cells have enough sterols and unsaturated
fatty acids and the mature forms of SREBP transcription
factors are not available. This sterol independent/cAMPdependent activation of CYP51 transcription might also be
important in somatic cells, such as liver, in some pathophysiological conditions. Understanding the CYP51 gene
transcription is important from the standpoint of its specific position in the cholesterol biosynthetic pathway. A better understanding of CYP51 transcription will help to a better understanding of the tissue-specific modes of cholesterol homeostasis.
FYS09 STRUCTURAL GENOMICS
OF CYP COMPLEMENT FROM
STREPTOMYCES:
APPLICATIONS FOR
GENERATING NEW
ANTIBIOTICS
a
b
synthase gene clusters. Thire activities include site-specific
oxidation of macrolide antibiotics precursors introducing
regiochemical diversity into the macrolide ring system,
thereby significantly affecting antibiotic activity. Efficient
manipulation of Streptomyces CYPs in generating new antibiotics will require identification and/or engineering of
monooxygenases with activities toward a diverse array of
chemical substrates. To better understand relation between
structure and function of CYP monooxygenases from secondary metabolic pathways of industrially important
Streptomyces, we have initiated the complete genetic, biochemical and structural evaluation of all 18 CYPs from
Streptomyces coelicolor A3(2). X-ray structures for two
S. coelicolor A3(2) CYPs assigned to the same CYP family, CYP154A1 and CYP154C1, have being determined.
Both structures are analyzed in context with other available
structures of related CYPs from polyketide synthase (EryF)
and non-ribosomal peptide synthetase (OxyB) biosynthetic
pathways. The generation of 3D-structures for evolutionarily related CYPs having the ability to hydroxylate different intermediates in macrolide antibiotic biosynthetic
pathways leads to an opportunity to translate patterns of
CYP amino acid sequences into structural patterns of folded proteins, a necessary step on the way to the rational design and modeling of CYPs of biotechnological as well as
physiological importance.
FYS10 INACTIVATION OF P450 2B1
AND 2B2 BY TERT-BUTYL
1-METHYL-2-PROPYNYL ETHER
c
L. M. PODUST , D. C. LAMB , H. BACH , and
M. R. WATERMANa
a
Department of Biochemistry, Vanderbilt University School
of Medicine, Nashville, Tennessee, 37232-0146, USA,
b
Wolfson Laboratory of P450 Biodiversity, Institute of
Biological Sciences, University of Wales Aberystwyth,
Aberystwyth, Wales SY23 3DA, UK, cDepartment of Microbiology and BioTechnology Institute, University of Minnesota, Minneapolis, Minnesota 55455, USA
[email protected]
The genus Streptomyces produces approximately twothirds of naturally occurring antibiotics as well as a wide
array of other secondary metabolites. Polyketides form one
of the largest and most diverse group of these natural products. They include numerous medicinal compounds e.g.
antibacterial, antifungal, anticancer and immunosuppressant agents. Although diverse in their structures, polyketide molecules share common features in their biosynthetic
pathways. Antibiotic diversity of polyketides is generated
during their biosynthesis by several means, including postpolyketide modification performed mainly by group transferases and oxidoreductases, very broad group of enzymes, which among others includes cytochrome P450 monooxygenases (CYP). CYPs are common in polyketide
S205
LINDA B. VON WEYMARNa,
and PAUL F. HOLLENBERGa
a
Department of Pharmacology, University of Michigan,
1150 West Medical Center Drive, Ann Arbor, Michigan,
48109, [email protected].
Acetylenic compounds can inactivate P450 enzymes
through binding of a reactive metabolite to either the apoprotein or the heme moiety. tert-Butyl 1-methyl-2-propynyl ether (tBMP) was tested for its ability to inactivate the
rat P450 enzymes 2B1 and 2B2. P450 2B1 and 2B2 are
97% identical, differing by only 14 amino acids. P450 2B1
and 2B2 have similar substrate specificity and exhibit similar regioselectivity for the metabolism of most substrates; however, the rate of substrate metabolism by 2B2 is
generally 5 to 10-fold lower than that of 2B1. tBMP inactivates the 7-ethoxy-4-(trifluoromethyl) coumarin (7-EFC)
O-deethylation activity of P450 2B1 in a time-, concentration-, and NADPH dependent manner; however, no inactivation was observed with 2B2. The loss of 2B1 activity
was accompanied by a loss in the reduced CO spectrum
and the amount of native heme with the concomitant appe-
arance of two heme adducts. LC/MS analysis of the heme
adducts resulted in an m/z of 705 for both adducts which is
consistent with an iron-depleted heme and a tBMP molecule containing one oxygen atom. The loss in 7-EFC O-deethylation (75%) was always greater than the loss in the
CO spectrum (31%) and native heme (39%). The ability of
tBMP to inactivate P450 2B1 and 2B2 mediated benzphetamine N-demethylation was also determined. Inactivation
of P450 2B1 by 10 µM tBMP resulted in a 22% decrease in
benzphetamine N-demethylation which is significantly less
than the 75% loss of 7-EFC O-deethylation that was observed. tBMP did not have an effect on P450 2B2 mediated
benzphetamine N-demethylation and no loss in either the
reduced CO spectrum or native heme was observed. tBMP
is a mechanism based inactivator of P450 2B1. In contrast
to P450 2B1, 2B2 is not inactivated by tBMP. These results
indicate that the relatively minor differences in the primary
sequence between these two isoforms have a dramatic
effect on the active site architecture.
Supported by NCI grant CA 16954.
FYS11 METABOLISM OF 1,8-CINEOLE
IN THE HUMAN LIVER AND
IDENTIFICATION OF THE
COMPETENT CYPS
MIKE DUISKENa, FRANK SANDNERa,
BRUNHILDE BLOEMEKEb,
and JULIANE HOLLENDERa
a
Institute of Hygiene and Environmental Medicine, RWTH
Aachen, Pauwelsstr. 30, 52074 Aachen, Germany, bDepartment of Dermatology and Allergology, University Hospital
RWTH, Pauwelsstr. 30, 52074 Aachen, Germany,
Among numerous groups of naturally occurring compounds examined so far, monoterpenes are known as fragrances and flavouring agents for example. 1,8-cineole,
a monoterpene cyclic ether which is named eucalyptol, is
found in essential oils like Eucalyptus polybractea. It has
a characteristic fresh fragrance and pungent taste and is extensively used for external application in pharmaceutical
preparations, e.g. in nasal spray or as disinfectant 1. The
metabolism of 1,8-cineole by liver microsomes of rats, humans and by recombinant cytochrome P450 enzymes was
investigated by Miyazawa et al.2 The results suggested that
1,8-cineole is solely oxidized in high rates to 2-hydroxy1,8-cineole and there was detected no other oxidation product.
In order to determine the toxic potential of monoterpenes we studied the human metabolism of monoterpenes in
vitro and in vivo. We investigate the biotransformation of
1,8-cineole in vitro by pooled human liver S9 (Figure 1)
and by recombinant cytochrom P450 3A4 and 3A5 enzy-
S206
Fig. 1. GC-MS-chromatograms with spektra of a sample after incubation without cytochrom P450 enzymes (2) and after incubatioon with human liver S9 (1). a) 2-hydroxy-1,8-cineole; b) 3-hydroxy-1,8cineole; c) 1,8-cineole
mes coexpressed with human CYP-reductase in Escherichia coli cells. Beside the product mentioned above, 2-hydroxy-1,8-cineole, we found one more metabolite produced
at high rates. It was identified by its mass spectrum as
3-hydroxy-1,8-cineole. There was a clear correlation between metabolite concentration and time of incubation and
enzyme concentration, respectively. Additionally, we
found different transformation rates of 2-hydroxy-1,8-cineole and 3-hxdroxy-1,8-cineole dependent on the cytochrome P450 3A enzyme used for incubation. In accordance to our results we detected after enzymatic hydrolysis
and sample preparation3 both metabolites in human urine
after oral administration of cold medication.
REFENCES:
1. Miyazawa, M.K.H., Morinaga K., Negoro K., Mura N.:
J Agric Food Chem, 37, 222 (1989).
2. Miyazawa M., Shindo M., Shimada T.: Drug Metab
Dispos, 29, 200 (2001).
3. Sandner, F., et al.: J. Chromatogr. B, 780, 225 (2002).
FYS12 CLEARANCE OF ANTIPYRINE
IN SALIVA OF LUNG CANCER
PATIENTS UNDER
IRRADIATION THERAPY.
ANDREY L. SUKHODUBa,
ALEXANDRA KIROCHKINAb,
and VIKTOR STARENKIYc
a
University of Portsmouth, UK, bKarazin National University and cInstitute of Medical Radiology, Ukraine.
Antipyrine clearance in body fluids is a recognized in
vivo indicator for the function of liver monooxigenase system. It is a well-known fact, that the development of tumours
is usually associated with the induction of certain CYP isoforms both in cancered tissie and in liver. We examined the
clearance of antipyrine (Cl) in saliva of lung cancer patients
(males 50-60 years old) before and after a therapy which was
a course of irradiation in intensive mode. It is established,
that Cl was apparently increased in the saliva of lung cancer
patients in comparison with control group of volunteers of
the same age and gender (6.04 versus 4.38 ml/kg/min). Consequently, half-time of antipyrine elimination (T1/2) in the
saliva of patients was decreased (5.9 versus 8 hours in control) and the apparent volume of distribution (Vd ) was increased (3.72 versus 2.88 l/kg in control group).
After the 2 week course of intensive radiation therapy
the drop in Cl (down to 2.62 ml/kg/min), increase in T1/2
(up to 21.9 hours) as well as normalisation of Vd (2.8 l/kg)
were observed in saliva of lung cancer patients.
It is concluded that enhanced antipyrine clearance in
saliva of lung cancer patients reflects activation of its excretion as a result of induction of certain isoforms of cytochrome P-450 participating in antipyrine biotransformation
process. The course of intensive therapy apparently inhibits the synthesis of CYP’s de novo and slows down antipyrine elimination from organism.
have cloned and expressed several of the P450 systems
from Mtb, along with redox partner enzymes - most notably the adrenodoxin reductase homologue FprA (encoded
by gene Rv3106) and the ferredoxin located adjacent to
Mtb’s sterol demethylase P450 (CYP51) on the chromosome (encoded by Rv0763c).
Kinetic characterization of the flavoprotein FprA reveal
intriguing features as regards interaction with pyridine nucleotide cofactors. While flavin (FAD) reduction rate shows
„regular“ hyperbolic dependence on concentration of
NADH reductant (Kd = 42.9 ± 4.6 µM, kred = 25.4 ± 0.7 s-1),
the situation is starkly different with NADPH (the tighterbinding coenzyme). Using stopped-flow methods, the FAD
reduction rate was shown to be reciprocally related to
[NADPH] at concentrations of the coenzyme (≤100 µM. The
reduction rate is constant at 20 ± 2 s-1 at [NADPH] above 100
µM, but accelerates dramatically at lower concentrations, e.g.
to 122.8 s-1 at 6.25 µM NADPH1. We consider that the phenomenon reflects dual binding sites for NADPH coenzyme,
as observed previously in studies of human CPR enzyme2.
The first site is catalytic, but the second (weaker) site is inhibitory, acting primarily to prevent release of NADP+ from the
catalytic site. FprA exhibits the most dramatic example yet
seen of this complex regulatory behaviour.
Ongoing studies on the Rv0763c-encoded ferredoxin
(Fer) reveal it to have a considerably more positive reduction potential than its primary redox partner (CYP51). Des-
FYS13 CYTOCHROME P450 REDOX
SYSTEMS IN MYCOBACTERIUM
TUBERCULOSIS
KIRSTY J. MCLEAN, DAVID LEYS,
KER R. MARSHALL, GERAINT LEWIS, ASHLEY J.
WARMAN, NIGEL S. SCRUTTON,
and ANDREW W. MUNRO
Department of Biochemistry, University of Leicester, University Road, Leicester, UK LE1 7RH. [email protected]
The genome sequence of the pathogen Mycobacterium
tuberculosis (Mtb) contains 20 distinct cytochrome P450
systems, an unprecedented number for a prokaryote. We
S207
Fig. 1. Atomic structure of CYP121 at 1.06 Å resolution. The heme
(in space fill) is sandwiched between the two major domains of the
structure: an α-helix-rich domain (dark gray) and the smaller _sheet-rich domain (light gray). The large water-filled active site
cavity of the enzyme is shown as a transparent surface. An arrow
indicates the possible site of substrate entry between helices F
and G.
pite this, turnover of CYP51 occurs in the presence of Fer
and an exogenous ferredoxin reductase3. This appears to be
a rare example of uphill electron transfer in a redox system,
perhaps reflecting a requirement to control carefully the
turnover rate of the CYP51 (and possibly other Mtb P450
partners for Fer).
In recent work, we have determined the high resolution
structure of a key Mtb P450 - CYP121 (encoded by the
Rv2276 gene) (Figure 1). The structure (at 1.06 Å) reveals
several novel features of cytochrome P4504. The heme cofactor is bound in two distinct conformations (related to
each other by a 1800 rotation about the CHα-Fe-CHδ axis of
the macrocycle), and is „kinked“ by an angle of approx. 300
at one of the pyrrole rings, due to interaction with the sidechain of Pro346. Several other important residues are immediately obvious, including the hydrogen-bonded Ser237
and Arg386, which dominate local structure above the
heme iron. Two distinct pathways (involving amino acid
residues and water molecules) by which protons can be relayed from the protein surface to the heme iron are evident.
These converge at Ser279, which exists in two different
conformations. In each of these positions it contacts one or
other of the pathways to the surface, and in both positions
it is able to pass protons to Arg386 (and on to the heme).
Importantly, studies of drug-binding to CYP121 demonstrate extremely high affinity for several azole drugs, particularly econazole and clotrimazole5. The nanomolar Kd
values for these drugs correlate well with MIC values for
their growth inhibitory action against Mycobacterium
smegmatis, providing strong evidence that the potency of
these drugs against mycobacteria derives from their efficient inhibition of CYP121.
REFERENCES
1. McLean K.J., Scrutton N.S., Munro A.W.: Biochem. J.
(in press) (2003).
2. Gutierrez A., Lian L.-Y., Wolf C.R., Scrutton N.S., Roberts G.C.K.: Biochemistry 40, 1964 (2001).
3. Bellamine A., Mangla A.T., Nes W.D., Waterman M.R.:
Proc. Natl. Acad. Sci. USA 96,8937 (1999).
4. Leys D., Mowat C.G., McLean K.J., Richmond A.,
Chapman S.K., Walkinshaw M.D., Munro A.W.: J. Biol.
Chem. 278, 5141 (2003).
5. McLean K.J., Cheesman M.R., Rivers S.L., Richmond
A., Leys D., Chapman S.K., Reid G.A., Price N.C.,
Kelly S.M., Clarkson J., Smith W.E., Munro A.W.: J.
Inorg. Biochem. 91, 527 (2002).
6. McLean K. J., Marshall K. R., Richmond A., Hunter I.
S., Fowler K., Kieser T., Gurcha S. S., Besra G. S.,
Munro A. W.: Microbiology 148, 2837 (2002).
S208
FYS14 ROLE OF CYTOCHROME P450
2E1 IN ETHANOL OXIDATION
IN THE BRAIN:
DETERMINATION WITH
KNOCK-OUT MODELS AND
INHIBITOR ANALYSIS
SIARHEY PRONKOa, SIARHEY ZIMATKINa,
PAVEL PRONKOb, and RICHARD DEITRICHc
a
Grodno State Medical University, Belarus, [email protected];
Institute of Biochemistry, Grodno, Belarus; cHealth Sciences
Center University of Colorado, Denver, USA
b
In contrast to the role of catalase, the role of CYP2E1 in
ethanol metabolism in the brain is controversial1,2,3,4. But recent studies demonstrated constitutive expression of
P4502E1 in brain, its differential induction in rat brain regions by chronic ethanol treatment, and its topographic distribution in rat and human brain5. The purpose of the study was
to clarify the contribution of cytochrome P450 2E1 in oxidation of ethanol to acetaldehyde in the brain tissue. In the
present study we tested the capacity of brain homogenates to
oxidize ethanol through a catalase and cytochrome P450-dependent system using the genetic strains of acatalasemic
mice (IACAT), mice with genetically specified deficiency of
cytochrome P450 2E1 (ICYP2E1KO), double mutants with
genetic deficiency of both catalase and cytochrome P450
2E1 (F3DKO) and control mice (C3H). The accumulation of
ethanol-derived acetaldehyde in brain homogenates incubated for 30 min in the presence of 50 mM ethanol in IACAT
mice was 47% of control value (P<0.01), in ICYP2E1KO
91% and in F3DKO mice 24% (P<0.01). The level of ethanol oxidation by brain tissue in vitro was significantly lower
in F3DKO mice as compared to acatalasemic animals. Preincubation of homogenates with diallyl sulfide (CYP2E1 inhibitor) significantly reduced the acetaldehyde accumulation
in control animals 23% (P<0.05). These experiments suggest
that CYP2E1 as well as catalase can play an important role
in ethanol metabolism in the brain.
Supported by 1R21AA12284-01, KO5AA00093,
P50AA03527, RO1-AA12650.
REFERENCES
1. Zimatkin S.M., Deitrich R.A.: Addiction Biol. 2. 387
(1997).
2. Aragon C.M.G., Ragan F., Amit Z.: Biochem. Biopharmacol. 44. 93 (1992)..
3. Gill K., Menez J. F., Lucas D., Deitrich R. A.: Alcoholism
Clinical and Experimental Research. 16. 910 (1992).
4. Pronko S.P., Zimatkin S.M., Deitrich R.A.: Alcoholism
Clinical and Experimental research. 6. 185A (2002).
5. Upadhya SC, Tirumalai PS, Boyd MR, Mori T, Ravindranath V.: Arch Biochem Biophys. 373, 23 (2000).
FYS15 POLYHALOGENATED
COMPOUNDS OF APPROPRIATE
CONFIGURATION INTERACT
WITH MAMMALIAN OR
BACTERIAL CYP ENZYMES TO
INCREASE BILIRUBIN
OXIDATION IN VITRO.
Table 1. Effect of either 3,4,3’,4’-tetrachlorobiphenyl (3,4-TCB)
or 2,4,2’,4’-tetrachlorobiphenyl (2,4-TCB) on the rate of bilirubin
oxidation by „supersomes“ preparations containing CYP1A1 or
CYP1A2.
Enzyme
Addition
CYP1A1
Role of an uncoupled catalytic cycle
NICOLETTA PONSa, FRANCESCO DE MATTEISa,b
DMSO
Medical Pharmacology, University of Turin, Via P.Giuria
13, 10125 Turin, Italy
b
Birkbeck College, University of London, Malet Street,
London WC1 E 7HX, UK
Polyhalogenated compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin are associated with toxic Uroporphyria1
and also causes alleviation of jaundice in the Gunn rat2
(and references therein). These effects have been attributed
to a microsomal oxidation of uroporphyrinogen and bilirubin for which supportive evidence has been obtained in
vitro. CYP1A1 required planar PCBs for these oxidative
reactions while CYP2B required non planar PCBs. Instead
CYP1A2 was capable of oxidation in their absence3.
We have now used genetically expressed rat CYP1A enzymes and confirmed with pure CYP1A1 that increased bilirubin oxidation was caused by the addition of 3,4,3’,4’tetrachlorobiphenyl (3,4-TCB). In contrast 2,4,2’,4’-tetrachlorobiphenyl (2,4-TCB), the nonplanar isomer, was
almost inactive. CYP1A2 was more active than CYP1A1 at
oxidizing bilirubin in presence of NADPH alone and reacted to addition of 3,4-TCB with a depression rather than
a stimulation of bilirubin oxidation (Table 1).
We have also tested a bacterial enzyme, CYP102 (a generous gift from Prof. Gordon C.K. Roberts of the University of Leicester). Dodecanoic acid and its polyhalogenated
analogue (perfluorododecanoic acid) both stimulated
NADPH oxidation by CYP102, but only the perfluoro analogue stimulated markedly bilirubin oxidation. The analogue exhibited much greater potency than the normal substrate in stimulating NADPH and bilirubin oxidation and
also showed greater affinity for CYP102, as measured by
the binding constant, Ks. The Ks values obtained for the
substrate and halogenated analogue (230 µM and 2.2 µM)
were in fairly close agreement with the EC50 values (approx. 300 and 1.5 µM) obtained for the oxidation of NADPH
and bilirubin by the two compounds. This supports the
view that occupancy of the activesite of the CYP enzyme
by the halogenated substrate analogue is involved in the
oxidative effects we have studied.
The molar stoichiometry ratio between NADPH and O2
consumption was 1 in the case of the substrate, but approximated 2 with the perfluoro analogue, suggesting - in the
case of the latter compound - an uncoupled catalytic cycle
S209
34.9 ± 8 (4)
3,4-TCB
106.8 ± 11.5 (4)*
2,4-TCB
52.8 ± 5.6 (3)
CYP1A2
a
Rate of bilirubin oxidation
(pmol/min)
DMSO
129.7 ± 45.2 (3)**
3,4-TCB
59.2 ± 7.6 (3)
2,4-TCB
113 ± 31.7 (5)
*
P< 0.001, compared with corresponding values obtained after
adding either DMSO (control) or 2,4-TCB
**
P< 0.01, compared with DMSO values obtained with CYP1A1
Table 2. Effect of dodecanoic acid or of perfluorododecanoic acid
on the production of H2O2 by CYP102.
Addition (final conc.)
H2O2 formed
nmol/m % of NADPH
oxidized
Dodecanoic acid
(150 µM)
2.6 ± 0.97 (3)
-
Dodecanoic acid
(150 µM) + NADPH (120 µM)
4.7 ± 1.1 (3)
1.8
Perfluorododecanoic acid
(1.5 µM)
1.4 ± 0.2 (3)
-
3.7 ± 0.01 (3)
1.93
Perfluorododecanoic acid
(1.5 µM) + NADPH (120 µM)
ultimately leading to full reduction of O2 to water. In contrast, no significant difference was found between dodecanoic acid and perfluorododecanoic acid in the amount of
H2O2 produced on incubation of CYP102 in presence of
NADPH (Table 2). We conclude that halogenated substrate
analogues interact with different CYPs to increase production of oxidative species, probably by an uncoupling mechanism. A role of the ferryl-oxygen intermediate is suggested in the oxidation of biologically important molecules, with possible implications for the therapy of jaundice
and for toxic oxidative reactions, such as uroporphyria and
cancer.
REFERENCES
1. De Matteis F.: Clinics in Dermatology 16, 265 (1998).
2. Zaccaro C. et al.: Biochem Pharmacol 61, 843 (2001).
3. De Matteis F., Dawson S.J., Pons N., Pipino S.: Biochem Pharmacol 63, 615 (2002).
07 Index 11.6.2003 13:04 Stránka 210
Author Index
AUTHOR INDEX
The following list is based on the
Scientific Program of the Conference,
i. e., the indicated code pertains
chiefly to the code of a particular presentation (underlining indicating the
presenting author). In most cases, but
not always, this is the same as the
code of the corresponding abstract, the
differences being caused chiefly by
late submissions and similar changes.
The code FYSd was used to indicate
the „short outline“ contributions to the
Forum of Young Scientists.
Abécassis V.
WP08
Acerbi D.
WP22
Agabeyli R.
MP53
Ahn T.
WP56
Achramovitch T.
TP29
Aimová D.
TP06, TP55
Akhtar M. K.
TP36
Akhtar M.
TP11
Alekperov U.
MP53
Ali S.
MP40
Allaeys V.
MP67
Allen K. E.
TP01
Allorge D.
M02
Amichot M.
TP28
Amin F.
WP26
Amjadi M.
TP49
Anderson A.
TL11, TP54, TP56,
TP59, TP62
Andersson K.
ML11
Andersson T. B.
MP26, TP38
André F.
MP32
Andreesen J. R.
WP62
Anfossi P.
WP67
Annaert P.
MP63
Annalora A.
MP29
Antonovic L.
MP54
Anzenbacher P.
WL04, MP12,
MP13, TP26,
TP68, WP17,
WP18, WP37
Anzenbacherová E.
TP26, WL04,
WP17, WP37,
TP68
Archakov A. I.
ML14, ML16,
ML19, MP20,
TP12, WP33, WP34
Arinc E.
TP65
Arnold F. H.
FYS06
Arpiainen S.
MP55
Arscott D.
TP23
Arthaud L.
TP28
Ask K.
WP40
Asperger O.
MP41
Assénat E.
TL08
Atabekov I. G.
TP60
Atkins W. M.
WP27
Avadhani N. G.
WL19
Aviles P.
Azeva T. N.
WP66
TP20
Baba T.
TL09
Backes W. L.
WL03
Badeaux J.
MP61, MP62
Badger T. M.
MP61, MP62
Baer B. R.
MP03, TP57
Baguley B. C.
WP39
Bach H.
FYS09
Baliharová V.
MP66, TP66
Ballou D. P.
WP35
Band M.
MP40
Banerjee A.
ML17
Baranová J.
TP26, TP68
Barra A.-L.
MP17
Bartakova P.
WP02
Bartashevich E. V.
WP19
Basran J.
CL03
Batt A. M.
MP42, MP56, MP59
Baudry J.
MP39
Bazin R.
WP30
Beaudet M.-J.
TL11, TP56
Beaudet M.-J.
TP54
Belejová M.
MP43
Bell D. R.
TP27
Bell S. G.
WP03, ML07
Bender V. E.
MP57
Bene‰ M.
MP13
Benveniste I.
MP37
Berenbaum M. R.
MP39
Beretta S. A.
MP29
Bernhardt R.
WL01, WP28
Berry J.
WP07
Bertrand-Thiebault C. MP42, MP56
Beynon E.
WP07
Bhaskar B.
MP28
Biagini C.
MP05, MP57
Bierach S.
TP67
Bichet A.
WP28
Bischoff D.
ML08
Bister B.
ML08
Bjorkhem I.
MP25
Blaney F. E.
MP02
Blobaum A. L.
WP29
Bloemeke B.
FYS11
Bode N.
WP66
Boer J.
TP01
Bochkareva A.
MP08
Bonifacio A.
FYSd, MP18
Bonina T. A.
TP19
Bonner R.
TP49
Boobis A. R.
TP38, WP26
Borde F.
MP57
Bofiek-Dohalská L.
MP04, TP06,
TP69
Boucher J.-L.
WP40, WP47
Breant D.
M02
Breskvar K.
TP31, TP35
Bride J.-M.
TP28
Broly F.
MP02
Brown N.
WP65
S210
Brun-Barale A.
Bryson T. A.
Brzakovic B.
Bfiezinová A.
Buhler D. R.
Bulko T. V.
Burak I.
Burdan F.
Bure‰ J.
Bylund J.
TP28
TL04
WP53
TP06
TP65
TP12
TP29
TP03, TP05
WP24
WP64
Cai X.
TP22
Calcutt M. W.
TL01
Calderon P. B.
MP58, MP67
Campbell A. P.
WP27
Camus P.
WP40
Canny B.
MP16
Canová N.
WP42
Capdevila J. H.
WL18
Carrie D.
WL15
Casse L.
WP40
Cassio D.
MP57
Catania S.
WP68, WP69, FYSd
Cauffiez C.
MP02
Celier C.
MP05
âervenková K.
MP43, MP44,
MP45,MP46
Cesaro C.
TP41
Chabot G. G.
TP63
Champion P. M.
WL02
Chang C. K.
M01
Charuel C.
MP57
Chauret N.
TP30
Cheesman M. J.
MP03, TP57
Chen C.-S.
WL07
Chen X.
ML07, WP03
Cheng D.
WL03
Chervin J. C.
MP16
Cheung C.
WL10
Chevalier D.
MP02
Chevalier S.
MP57
Ching L.-M.
WP39
Chiu M. L.
MP29
Chiu R.
FYS02
Chladek J.
WP37
Chmela Z.
MP43, MP44,
MP45, MP46
Chougnet A.
TL07
Chowdry J.
M02
Chuang S.
TP32
Chudaev M. V.
TP21
Chui B.
TP49
Clarke S.
Coecke S.
Cohen S.
Coller J. K.
Commandeur J. N. M. MP18,
Contzen J.
Cosme J.
Costa C.
TP67
WL14
MP06
TP42
MP33,
FYS03
MP17
ML03
WP69, WP68
07 Index 11.6.2003 13:04 Stránka 211
Cotman M.
Crochard D.
Cruciani G.
Cui X.
Author Index
WL09
TP28
MP26
MP54
D’Angelo V.
WP69
Da Silva M. E. F.
TP43
Dabrowski M. J.
WP27
Dahl C.
MP61, MP62
Daniel W. A.
TP02, WP32, WP55
Dash P. K.
WL16
Dauvergne M.-T.
MP23
David Stout C.
FYS02
Davies D. S.
WP26
Davydov D. R.
MP07, MP08
Dawidek-Pietryka K.
TP04
Dawson J. H.
TL04
Day S.
TP30
De Coster R.
WP66
de Graaf C.
MP33, FYS03
de la Garza M.
MP27
De Matteis F.
FYS 15
de Visser S. P.
WP36, WP43
Debeljak N.
MP35
Deitrich R.
FYS14
Delaforge M.
MP32
Denisov I. G.
MP21, TL03
Desrochers M.
TP59
DeVoss J. J.
WP01
Didolkar V.
TP22
Dijols S.
WP40, WP47
Dilworth C.
WP04
Ding Q.
WP39
Dlouhá T.
TP06, TP55
Doddapaneni H.
TP51
Dodhia V.
WP30
DolÏan V.
TP31, TP35
Domaƒska K.
TP04
Don M.-J.
MP30
Dorokhov Y. L.
TP60
Drocourt L.
TL08
Droech S.
MP42
Drutsa V. L.
TP48
Duan H.
MP40
Dudka J.
TP03, TP04, TP05
Duisken M.
FYS11
Dunn A. R.
FYS02
Durst F.
MP37
Eagling V.
Ebner R.
Edom R. W.
Edwards R. J.
Ehlers A. W.
Eice B. C.
Eichelbaum M.
Eldarov M. A.
Ellis S. W.
El-Nezami H.
Endrizzi K.
WP04
MP36
TP22
TP38
MP34
WP63
MP50, TP10,
TP37, TP41, TP52
TP60
MP02
WP02
MP50
Ericksen S. S.
Essigke T.
Estabrook R. W.
Evans D. C.
Evers R.
Eya M.
MP09
ML17
TP19
TP22
TP22
TP61
Fairhead M. J.
Fantuzzi A.
Farghali H.
Farinas E. T.
Feenstra K. A.
Feldman R.
Fenn P. D.
Ferguson M.
Ferhatoglu Y.
Ferrari L.
Fiala Z.
Fields P. R.
Filipec M.
Fink M.
Finn R.
Fischer V.
Flanagan J. U.
Fon Tacer K.
Forsterová K.
Frapart Y.
Frei E.
WP05
MP10
WP42, WP50
FYS06
MP33
TP49
WP04
MP61, MP62
MP40
MP42, MP56, MP59
WP18
WP44
WP50
FYS08, WL09
CL03
TP24
MP11
TP58, WL09
TP06, WP20
WP40, WP47
TP06, TP55,
WL11, WP16,
WP20, WP23
Froenzler D.
MP42
Fuhr U.
WL05, TP45
Fujii-Kuriyama Y.
TL09
Fujino H.
WP31
Fukushige S.
WP10
Funae Y.
MP47
Furge L. L.
WP44
Garcia C.
MP28
Gasimova T.
MP53
Gaskin M.
TP49
Georgescu O.
FYSd
Gerbal-Chaloin S.
TL08
Gilardi G.
MP10, WP05, WP30
Gilep A. A.
TP17, TP18, TP19
Gillam E. M. J.
WP01, TP57
Gillis D.
TP42
Giroud C.
WP40
Glieder A.
FYS06
Globokar T.
TP35
Gonzalez F. J.
WL10
Goode W. E.
WP44
Goodin D.
B.FYS02
Gooijer C.
MP18
Gorren T.
ML11
Graham S.
MP25, WP65
Granvil C. P.
WL10
Gravel M.
TL11, TP59
Gray H. B.
FYS02
Grinstead J. S.
WP27
Grishanova A. Y.
MP64
S211
Grishina M. A.
WP19
Grivennikov S. I.
TP47
Groenhof A. R.
MP34
Grubor V.
MP48
Gruenke L.
TP23
Guan Z.-W.
FYS04, WP45, WP51
Guengerich F. P.
TL01, WP57
Guillouzo A.
WL12
Gusev S.
ML14
Gut I.
TP50, WP54
Gutierrez A.
CL03
Gwynne P.
TP49
Hada‰ová E.
Haduch A.
Haertlein M.
Hahn U.
Hakkola J.
Haley R.
Halpert J. R.
Han S.-M.
Hanna I. H.
Hannemann F.
Harada H.
Hardy H.
Harisson D.
Harlow J.
Harris D. E.
Harris D.
Hays A.-M. A.
Hazai E.
He Y. A.
He Y. Q.
Heck M.
Heckel D. G.
Heinkele G.
Helvig C.
Henderson C. J.
Hendrickx J.
Hengstler J.-G.
Hering U.
Herman D.
Hidestrand M.
Hirano M.
Hirata T.
Hiroi T.
Hodek P.
Hodis J.
Hof M.
Hofte B.
Holãapek M.
Holla V.
Hollenberg P. F.
Hollenberg P. F.
Hollender J.
Horley N. J.
Horsk˘ S.
Houle R.
Howard L.
WP41
WP32
MP23
MP41
MP55, TP44
MP61, MP62
ML01, ML02,
MP07, MP08
WP61
TL01
WP28
TP13
MP61, MP62
MP61, MP62
M02
ML09
MP27
FYS02
TP08
ML02
ML01
ML08
MP48
MP50
TP32, TP33, TP34
WL15
MP63, WP66
WL13
TP45
TP35
MP60
WP31
MP19
MP47
MP04, MP68,
TP09, WP70,
WP11, TP69, TP70
FYSd
MP13
TP07
WP17
WL18
FYS10
WP29, WP35
FYS11
TP27
WP54
TP30
WL17
07 Index 11.6.2003 13:04 Stránka 212
Author Index
Hudeãek J.
MP12, MP13
Hugenschmidt S.
MP49
Hughes K. T.
WP07
Hui Bon Hoa G. MP14, MP16, WP34
Humphrey L.
MP61, MP62
Hurkmans R.
WP66
Idle J. R.
WL10
Igarashi T.
MP47
Ignatovets O.
TP29
Imaoka S.
MP47
Ingelman-Sundberg A. CL04, MP60,
TP38, TP45
Iscan M.
WP63
Ishibashi T.
WP15
Ishimori K.
TP13
Ishimura Y.
ML09, WL11, WP06
I‰tvánková B.
TP09
Ito A.
TP18
Itoh S.
MP52
Ivanov A.
ML16
Ivanov Yu. D. WP33, WP34, MP20
Iwanari M.
MP52
Iyanagi T.
FYS04, TP52, WP45,
WP51
Izumi S.
MP19
Jabrane W.
TP45
Jackson C.
ML18, TP40
Jacobs D.
MP63
Jáchymová M. WP06, WP46, ML09
Jalonen J.
TP15
James H. M.
TP42
Jauregui J.
WP25
Jetter A.
TP45
Jgoun A. A.
TP60
Jhoti H.
ML03
Jiang T.
TP27
Jin S.
TL04
Jodynis-Liebert J.
TP03, TP05
Johansson A.
TP38
Johansson I.
TP38
Johnson E. F.
ML02, ML04
Jones G.
TP32
Jounaidi Y.
WL07
Jourdan N.
MP15
Jung C.
MP16, MP17
Juvonen R. O. WP02, WP21, MP31
Kaderbhai M. A.
Kaderbhai N. N.
Kaloshyna N.
Kalsotra A.
Kanaeva I. P.
Kaneko H.
Karabec M.
Karlgren M.
Karuzina I. I.
Kasel D.
Kasper C.
TP36
TP36
WP09
WL16
WP34, WP33
MP19
W70
TP45
WP34
TP45
TP23
Kato S.
TL09
Katsiabas D. S.
WP59
Kawashima H.
MP54
Keizers P. H. J.
MP18, FYS03
Kelley R. W.
WL03
Kelly D.
ML18, TP40
Kelly S. L.
CL02
Kelly S.
ML18, TP40
Kemp C.
MP11
Kent U. M.
WP29, WP35
Kestell P.
WP39
Khomich T.
WP09
Kiessling A.
MP36
Kikuchi H.
WP10
Kikuta Y.
TP61
Kim C.-J.
WP61
Kim E.-Y.
WP57
Kim J.-S.
WP57
Kim J.-W.
WP61
Kim K.-A.
WP60, WP61
Kim K.-H.
WP56
Kim M.-Y.
WP57
Kimura S.
TP53, WP45, WP51,
FYS04
Kinzig-Schippers M.
TP45
Kirikbakan A.
TP64, TP65
Kirochkina A.
FYS12
Kishimoto W.
MP47
Kitagawa T.
MP12
Kitazume T.
WP06
Klein K.
TP37, TP10,
TP41, TP52
Klepacz R.
TP03, TP05
Kmoníãková E.
WP42
Koblas T.
MP68
Koh W.-S.
WP57
Kojima J.
WP31
Kokhanovski V. V.
TP17
Koláfiová L.
WP17
Komel R.
TP39
Kominami S.
MP19
Konas D.
WP49
Kondrová E.
WP12
Korczak B.
TP32, TP33, TP34
Kordi‰ D.
MP35
Kores-Plesniãar B.
TP31
Kot M.
TP02
Kousalová L.
TP68, WL04
Kovalenko V.
MP38
Kovaleva I. E.
TP47
Král R.
TP14
Krauser J. A.
TL01
Kuãka J.
TP06
Kukaãková K.
TP06
Kumar A.
TP07
Kumar D.
WP43
Kumar S.
ML01, MP08
Kumst˘fiová T.
WP23
Kunishi H.
TP61
Kunze K. L.
MP03, TP01
Kupfer A.
WL10
Kurchenko V. P.
TP21
Ku‰ãer E.
TP39
S212
Kuznetsov V. Yu.
MP20, WP33,
WP34
Kvasniãková E.
TP14
Kvûtina J.
WP17, WP18, WP24
Kwon J.-T.
WP58
Lah L.
Lachaud A.
Lai T.-S.
Laizier M.
Lamb D. C.
TP39
TL11, TP54
M01
TL11, TP62
CL02, ML18, TP40,
FYS09
Lambert H.
MP59
Lamczyk M. C.
TL04
Lamka J.
MP66, TP66
Lammertsma K.
MP34
Lampe J. N.
WP27
Lang T.
TP37, TP52
Lange R.
ML11
Langouët S.
WL12
LaÀková M.
WP23
Lanza D. L.
TP25
Larrey D.
TL08
Lazar A.
TP45
Ledesma A.
TP49
Ledger R.
TP07
Lee J.-S.
WP60, WP61
Lee-Robichaud P.
TP11, WP38
Lefevre D.
WP47
Lehnert A.
TP67
Lei L.
CL02
Leifels T.
TL06
Lemr K.
MP45, MP46
Lendzian F.
MP17
Leont’ev V.
TP29
Lepesheva G. I.
TP20, WL08
Lewis D. F. V.
MP30
Lewis G.
FYS13
Leys D.
FYS13, WP13
Lhermitte M.
M02
Li H.
ML06, MP24, MP28
Li X.
MP39
Liddle C.
TP67
Lim S.
WP60, WP61
Lin H.
MP06
Lisitsa A.
ML14, ML16, ML19
Lo-Guidice J.-M.
MP02
Loiseau N.
MP32
Lonhienne T. G. A.
WP01
Lu H.
WL07
Lück H.
TP45
Ludewig M.
MP41
Lupp A.
MP49
Luzikov V. N.
TP47, TP48
Makris T. M.
MP21, TL03
Malaplate-Armand C.
MP59
Malmebo S.
TP38
Mancy A.
MP15
Mannens G.
MP63, WP66
Mansuy D.
ML10, WP40, WP47
07 Index 11.6.2003 13:04 Stránka 213
Manzanares I.
WP66
Marangoni D.
TP43
Marczylo T.
TP40
Marchal S.
ML11
Marie B.
MP42
Marill J.
TP63
Marková V.
WP11, WP23
Maroun R. C.
MP05
Marshall K. R.
FYS13, WP13
Martásek P.
ML09, WP06, WP46
Martin M. V.
TL01
Martínek J.
WP50
Martínek V.
WP11, WP14
Martínková J.
WL04, WP37
Martino L. B.
WP68, WP69
Martinovic Î.
WP53
Martins D.
TP43
Marvasi L.
WP67
Marx C.
MP50
Masson C.
MP56, MP59
Masters B. S. S.
ML09, MP27,
WP06 WP46
Masumoto Y.
TP61
Matayoshi E. D.
MP29
Maton N.
MP67
Matsui K.
WP15
Maurel P.
TL08, TL10, WP55
Maurer S. C.
TP16
Mayer B.
ML11
McLain J.
ML09
McLaren A.
WL15
McLaughlin L.A.
MP22
McLean K. J. CL03, WP13, FYS13
Meilleur F.
MP23
Meinhold P.
FYS06
Meirelles N. C.
TP43
Meuldermans W.
MP63, WP66
Meyer D.
TL07
Micuda S.
WP37
Miãáková A.
TP70
Miksys S.
WL17
Mik‰anová M.
WP16
Miljkovic B.
WP53
Miller G. P.
TL01
Miller W. L.
TP48
Mimura J.
TL09
Miroshnichenko Y.
ML14, ML19
Mitani F.
WL11
Mock M.
MP27
Moguilevsky N.
MP03
Mohanty S.
FYS06
Monostory K.
TP08
Moreau M.
WP47
Morel F.
WL12
Morgenstern R.
MP37
Morishima I.
TL05, TP13
Morohashi K.
TL09
Motohashi H.
TL09
Mukai K.
WL11
Müller D.
MP49
Müller M.
TL07
Mundlova L.
WP37
Munro A. W.
CL03, WP13,
Author Index
Mürdter T. E.
Murias M.
Murray G. R.
Murtazina D.
Myles D.
MP12, FYS05, FYS13
MP50, TP10
TP03, TP05
WP04
MP25
MP23
Nádvorníková R.
WP18
Nagano S.
ML06, MP24, TP13
Nakajima M.
MP52, WP58
Nakata K.
TP61
Narasimhulu S.
CL07
Nazarov P. A.
TP47, TP48
Negishi M.
TP44
Nicoll-Griffith D.
TP30
Nishida C.
ML06, MP24
Nishii S.
WP15
Nishimoto S.
TP61
Nishimura K.
WP58
Nishino Y.
WP45, WP51, FYS04
Nobilis M.
WP17
Novello J.C.
TP43
Novikov V.
K TP60
Novikova L. A.
TP47, TP48
Numayama-Tsuruta K.
TL09
O’Donnell E.
WP38, FYSd
Oesch F.
WL13
Ogura H.
ML06, MP24
Ohkawa H.
CL06
Ohta J.
TP61
Ohtake F.
TL09
Oka M.
WP15
Oliw E. H.
WP64
Omdahl J. L.
MP29
Ortiz de Montellano P. R.
ML05,
ML06, MP24,
WP52, FYS01
Osada M.
MP47
Otter C.
TP38
Otto D.
WL15
Ozdamar E. D.
WP63
Pääbo S.
Paine M. J. I.
Palamara A.
Palmer B. D.
Pan L.
Panda S. P.
Pandova B.
Pappas I. S.
Paquet Y.
Park H.-J.
Park J.-Y.
Parker J.
Pascual L.
Pascussi J. M.
Pastera J.
Pastuszyn A.
Pávek P.
MP41
CL03, MP11, MP22
WP68
WP39
MP39
ML09, WP06, WP46
WP52
WP59
TL11, TP62
WP61
WP60
ML18, TP40
WP35
TL10, TL08
WP18
MP29
TP14
S213
Paxton J.
Pearson J. T.
Peck T.
Pekárek J.
Pelkonen O.
WP39
WP27
TP49
MP13
MP31, MP55,
TP15, TP44, WL06,
Pelzer S.
ML08
Perally S.
TP40
Peternel P.
TP35
Peters M. W.
FYS06
Peters T.
TP49
Peterson J. A. MP25, WP06, ML15
Petkovich M.
TP32, TP33
Petushkova N. A.
TP60, WP33
Philpot R.
MP05
Picard-Cloutier A.
TP63
Pichard-Garcia L.
TL08, WP55
Pikuleva I.
MP25
Pl‰ková J.
WP50
Podust L. M.
FYS09, WL08
Pogrebnoy A. A.
WP19
Pokrajac M.
WP53
Poljaková J.
TP06, WP20
Pompon D.
WP08
Ponomarenko E.
ML19
Pons N.
FYS15
Popl‰tein M.
TP70
Poso A.
WP21
Potemkin V. A.
WP19
Poulos T. L.
ML06, MP24,
MP28, TP13
Price-Jones M.
WP07
Pronko P.
FYS14, WP09
Pronko S.
FYS14
Provazníková D.
TP09
Psotová J.
TP68
Puccini P.
WP22
Pylypenko O.
ML08
Rahnasto M.
WP21
Raimundo S.
TP41
Raman C. S.
ML13
Rane A.
MP60
Rao Z.
ML07, WP03
Raunio H.
WP02, WP21
Ravãuková B.
WP41
Reeves M.
MP61,
Reilly C. A.
TP25
Rettie A. E.
MP03, TP57
ReÏen T.
MP35
Ricard D.
TL07
Riccardi B.
WP22
Ridderström M.
MP26
Rieber E. P.
MP36
Rieger M.
MP36
Riegrová D.
MP43, MP45, MP46
Richter T.
TP10, TP37, TP52
Rivory L.
TP67
Roberts G. C. K. CL03, MP11, MP22
Robertson G.
TP67
Robinson J. A.
ML08
Rodríguez-Antona C.
MP60
07 Index 11.6.2003 13:04 Stránka 214
Author Index
Rohayem J.
Roitel O.
Roman L. J.
MP36
CL03, FYS05
ML09, MP27, WP06,
WP46
Ronis M. J. J.
MP61, MP62
Roof R. A.
WP35
Rosewell I.
WL15
Rosic N.
WP01
Rousett S.
ML08
Roymans D.
MP63
Rozman D.
MP35, TP58, WL09,
FYS08
Ryabinin V. E.
WP19
Rydberg P.
WP48
Ryde U.
WP48
Rypka M.
MP43, MP44,
MP45, MP46
Sagami I.
·alandová J.
Sallustio B. C.
Sandner F.
Santolini J.
Sari M.-A.
·armanová J.
Sato H.
Sato Y.
Sbaragli L.
Schaeffeler E.
Schänzle G.
Schauer L.
Schlichting I.
ML12
WP18
TP42
FYS11
WP49
WP40
TP50
TL01
ML12
TL06
TP41
MP50
WP64
ML08, MP21,
MP23, TL03
Schmeiser H. H.
WP16, WP23
Schmid R. D.
TP16
Schmitz M.
MP36
Schoedel K.
WL17
Schoch G.
ML04
Schöneboom J. C.
MP06
Schuler M. A.
MP39, MP40
Schünemann V.
MP17
Schwab M.
MP50, TP10,
TP37, TP41, TP52
Schwaneberg U.
FYS07
Schwartz P. S.
WL07
Schwarz C.
MP41
Scott E. E.
ML01, ML02
Scrutton N. S. CL03, FYS05, FYS13
Sejbal J.
TP06
Sen A.
TP64, TP65
Sergeev G. V.
TP18
Sevrioukova I.
ML06, MP28
Shaik S.
MP06, TL02,
WP36, WP43
Shannly-Henderson R.
WP04
Sharma P. K.
WP43
Shea T. M.
ML09, WP46
Sheikh Q.
TP11
Shen A.
TP23
Shibazaki M.
WP10
Shim I.-S.
WP61
Shimada S.
WP31
Shimizu H.
MP24
Shimizu T.
ML12, MP12
Shimoji M.
MP37
Shkumatov V. M.
TP48
Shumyantseva V. V.
TP12
Sidorova Y. A.
MP64
Sielaff B.
WP62
Siest G.
MP59
Sigfridsson E.
WP48
Silbergleit A.
TP49
Silva J. A.
TP43, TP30
Silvari V.
WP68, WP69
·i‰pera L.
MP65
Skálová L.
MP65, MP66, TP66
Skaug T.
ML18
Skordos K. W.
TP25
Skott A.
TP45
Skryabin K. G.
TP60
Skvortsov V.
ML16
Slaviero K.
TP67
Sleno L.
TP30
Sligar S. G.
MP21, WP34, TL03
Slíva J.
MP51
Smeal S. J.
TP25
Smrãek S.
TP09
Soerlie M.
ML11
Sogawa K.
TL09
Sohel A.
WP10
Somogyi A. A.
TP42
Sopko B.
WP23
Sopková K.
WP23
Sörgel F.
TP45
Souãek P.
TP26, TP50, WP12
Sowden R. J.
ML07
Spinabelli D.
WP22
Starenkiy V.
FYS12
Starikow J.
MP33
Stark K.
WP64
Stegnar M.
TP35
·tûrba M.
FYSd
Stiborová M.
MP04, MP68, WP11,
WP14, WP16, WP20,
WP23, TP06, TP09,
TP55, TP69, WL11
Stiborová-Rupertová M.
TP06
Stoessel C.
TL07
Stout C. D.
ML02, ML04
Straub W. E.
FYS01
Straub W.
ML05
Strobel H. W.
MP54, WL16
Strushkevich N. V.
TP20
Stfie‰tíková P.
WP50
Stuehr D. J.
WP49, WP40, WP47
Su T.
WL07
Suematsu M.
WL11
Sukhodub A. L.
FYS12
·ulc M.
TP69, W70, WP16, WP20
·Ûsová S.
TP50
Süssmuth R. D.
ML08
Sutcliffe M. J.
MP11, MP22
Svoboda Z.
WP24
Swart A. C.
WP65
S214
Swart M.
Swart P.
Szklarz G. D.
Szotáková B.
Szumi∏∏o J.
MP34
WP65
MP09
MP65, MP66, TP66
TP03, TP05
Taimi M.
Takahashi S.
Takeuchi T.
Tares S.
Tcherviakovsky E. M.
Temme A.
Tesafiová M.
Theisen M. J.
Thiel W.
Thompson A.
Tjaden U.
Todorova V.
Tomalik-Scharte D.
Tomandl J.
Tosha T.
Town M.
Tran P.
Trautwein A. X.
Trefil P.
Trejtnar F.
Tressler M. C.
Trottier É.
Truan G.
Truss R. S.
Tsai M.-W.
Tsuchiya Y.
Tsunenari Y.
Tucker G. T.
Turpeinen M.
Tyndale R. F.
Ueng Y.-F.
Uherková L.
Ullmann M.
Ulvila J.
Urban P.
Urlacher V.
Usanov S. A.
Uusitalo J.
TP32, TP33
TP13
WP10
TP28
TP21
MP36
FYSd
MP29
MP06
MP42
TP07
WP52
TP45
WP41
TP13
WP26
TP24
MP17
MP68, TP70
TP14
WP44
TP59
WP08
WP44
MP30
MP52
WP31
MP02
TP15, WL06
WL17
MP30
MP43
ML17
TP44
WP08
TP16
TP17, TP18,
TP19, TP20,
TP21, WP34, MP20
WL06, TP15
Václavíková R.
Valentová P.
van der Vies
van der Vies S. M.
van der Zwan G.
Van Houdt J.
Vannelli T. A.
Vazquez-Duhalt R.
Velickovic R.
Velík J.
Verbeeck R. K.
Vereczkey L.
WP54
TP69
FYS03
MP18
MP18
MP63, WP66
WP52
WP25
WP53
MP66, TP66
MP58, MP67
TP08
07 Index 11.6.2003 13:04 Stránka 215
Vermeir M.
Vermeulen
Vermeulen N. P. E.
Vesel˘ J.
Vianello R.
Viitala P.
Vilarem M. J.
Vinkovic D.
Visvikis S.
Vitali F.
Voegtle H. L.
von Weymarn L. B.
Vondrá‰ková L.
Voronina A.
Vofií‰ek V.
Vrijbloed J. W.
Wade R. C.
Walchner-Bonjean M.
Wang R. W.
Wang S.-Y.
Wang Z.-Q.
Ward A.
Warman A. J.
Warrilow A.
Waskell L.
Waterman M. R.
Wauthier V.
Waxman D. J.
Weigle B.
WP66
FYS03
MP18, MP33
MP46
MP26
MP31
TL10, TL08
ML03
MP42,
MP56, MP59
ML08
TL04
FYS10
WP16
MP38
WP24
ML08
ML17
TP45
TP22
MP30
WP49
ML03
FYS13
ML18
TP23
WL08, CL02,
WL18, FYS09
MP58, MP67
WL07
MP36
Author Index
Wen Z.
Werck-Reichhart D.
Wester M. R.
Westlind-Johnsson A.
Weygers A.
White J. A.
White M. A.
Wiessler M.
Willcox J. K.
Williams P.
Winkler J. R.
Winn P.
Woggon W.-D.
Wohlleben W.
Wójcikowski J.
Wolf C. R.
Wong L.-L.
Wong T. S.
Wright J. N.
Wsól V.
Xu F.
MP39
MP40
ML02, ML04
TP38
MP63
TP32
ML02
WP23
CL07
ML03
FYS02
ML17
TL06, TL07
ML08
TP02, WP55
CL03, MP11,
MP22, WL15
ML07, WP03
FYS07
WP38
MP65, MP66,
TP14, TP66
ML07, WP03
Yadav J. S.
Yamada I.
Yamamoto K.
TP51
WP31
WP45,
WP51, FYS04
TL09
TP49
Yamamoto M.
Yamashiro C.
S215
Yamazaki T.
Yanev S.
Yano J. K.
Yeung L.-L.
Yin H.
Yokoi T.
Yoshida R.
Yoshioka S.
Yost G. S.
Yu A.
Yun C.-H.
MP19
WP52
ML04, TP13
M01
TP24
MP52, WP58
WP58
TP13
TP25
WL10
WP56, WP57
Zaghini A.
Zahlsen K.
Zachová M.
Zalar B.
Zamora I.
Zanger U. M.
WP67
WP68, WP69
TP06
TP31
MP26
CL05, TP10,
TP37, TP41,
TP52, MP50
ML08
TP23
ML08
MP03
WP39
FYS14
WP41
TP26
TP52
WP66
Zerbe K.
Zhang H.
Zhang W.
Zheng Y.-M.
Zhou S.
Zimatkin S.
Îourková A.
Zuber R.
Zukunft J.
Zwijsen C.
07 Index 11.6.2003 13:04 Stránka 216
LIST OF REGISTERED
PARTICIPANTS
(as of June 6, 2003)
Abe Koji
Sankyo Co., Ltd.
Pharmacokinetics and Drug Delivery
Research Laboratories
1-2-58, Hiromachi, Shinagawa-ku
Tokyo
140-8710
Japan
Abernethy Lynn
Quintiles Ltd.
DMPK + Bioanalysis Research Av.
Research Park
Edinburg
EH14 4 AP
United Kingdom
Aimová Dagmar
Charles University
Biochemistry
Hlavova 2030
Prague 2
128 40
Czech Republic
Akhtar Mohammed Kalim
University of Wales
Inst. of Biological Sciences
Penglais
Aberstwyth
SY23 3DA
United Kingdom
Akhtar Masoud
University of Birmingham
Biosciences
Ring Road South
Birmingham
B15 2TT
United Kingdom
Akpan Charles
Transhumanist Association of Nigeria
Research
75 Ojuelegba Road
Surulere
23401
Nigeria
Allorge Delphine
Faculte de Medecine
EA2679
1 place de Verdun
Lille
59045
France
Amichot Marcel
INRA
UMR n°1112 -API lab.123, Bd. F. Meilland, BP 2078
Antibes cedex
6606
France
Amin Farhana
Imperial College London
Section on Clinical Pharmacology
De Cane Road
London
W12 ONN
United Kingdom
Anderson Alan
L’Hotel-Dieu de Québec
Centre de Recherche
11 Cote du Palais
Québec
G1K 7P4
Canada
Andócs Gábor
Sanofi Synthelabo
Pharmacokinetics
Tó Utca 5
Budapest
1045
Hungary
André François
CEA-CNRS
Dept of Biology Joliot-Curie
Service de Bioénergétique, URA
2096 CNRS, DBJC, CEA-Saclay
Gif-sur-Yvette cedex
91191
France
Antonovic Leposava
UT Medical School
Biochemistry and Molecular Biology
6431 Fannin
Houston
77030
Yoguslavia
Anzenbacherová Eva
Palacky University
Faculty of Medicine
Institut of Medical Chemistry
and Biochemistry
Hnûvotínská 3
Olomouc
775 15
Czech Republic
Anzenbacher Pavel
Palacky University
Faculty of Medicine
Pharmacology
S216
Hnûvotínská 3
Olomouc
775 15
Czech Republic
Archakov Alexander I.
Institute of Biomedical Chemistry
Russian Academy of Medical
Sciences
Pogodinskaya str., 10
Moscow
119121
Russia
Armand-Malaplate Catherine
Faculty of Pharmacy
Toxicology
30 rue Lionnois
Nancy
54000
France
Arpiainen Satu
University of Oulu
Pharmacology and Toxicology
Box 5000 (Aapistie 5)
Oulu
90014
Finland
Asperger Otmar
University of Leipzig
Faculty of Biosciences
Institute of Biochemistry
Brüderstr.34
Leipzig
D-04103
Germany
Avadhani Narayan
University of Pennyslvania
Animal Biology
3800 Spruce St.
Philadelphia
PA 19063
United States of America
Babetas John
Xanthus Life Sciences Inc.
Research and Development
225 President Kennedy Suite
PK-5660
Montreal, Quebec
H2X 3Y8
Canada
Backes Wayne
Louisiana State U.
Health Sciences Center
Pharmacology
533 Bolivar St.
New Orleans, LA
07 Index 11.6.2003 13:04 Stránka 217
70112
Backfisch Gisela
Abbott GmbH c Co. KG
GG PSE
Knollstr
Ludwigshafen
67061
Germany
Baer Brian
University of Washington
Department of Medicinal Chemistry
BOX 357610
Seattle
98195
Banerjee Amit
Wayne State University
Inst. of Env. Health Sciences
2727 Second Avenue, Rm. 4327
Detroit
48201
Baranova Jana
University of Palacky
Faculty of Medicine
Hnûvotínská 3
Olomouc
77515
Czech Republic
Bartáková Petra
Pharmaceutical Faculty
Charles University
Pharmacy
Heyrovského 1203
Hradec Králové
500 12
Czech Republic
Batt Anne-Marie
Faculty of Pharmacy
Centre du Medicament
30, rue Lionnois
Nancy
F 54000
France
Beaudet Marie-Josée
Centre de Recherche en Cancérologie
Centre de Recherche
11 Cote du Palais
Québec
G1K 7P4
Canada
Belejová Marie
Palacky University
Faculty
of Medicine
Pharmacology
Hnûvotínská 3
Olomouc
775 15
Czech Republic
Bonifacio Alois
Vrije Universiteit Amsterdam
Chemistry
De Boelelaan 1105
Amsterdam
1081 HV
Netherlands
Bell Stephen
Oxford University
Inorganic Chemistry Laboratory
South Parks Rd
Oxford
OX1 3QR
United Kingdom
Bofiek-Dohalská Lucie
Charles University
Biochemistry
Hlavova 2030
Prague 2
128 40
Czech Republic
Bell David
University of Nottingham
School of Life
University Park
Nottingham
NG72RD
United Kingdom
Boucher Jean-Luc
UMR8601 CNRS
45 rue des Saints Peres
Paris
75270
France
Bernhardt Rita
Saarland University
Dept. of Biochemistry
Postfach 151150
Saarbrucken
66041
Germany
Brun Barale Alexandra
INRA
UMR1112 Rose Team API
123 bv Francis Meilland
Antibes
6160
France
Bertrand-Thiebault Céline
INSERM U 525
30 rue Lionnois
Nancy
54000
France
Buc Calderon Pedro
Université Catholique de Louvain
Sciences Pharmaceutiques
73, avenue E. Mounier
Bruxelles
1200
Belgium
Biagini Christine
Pfizer
MCT
Z.I. Poce sur Cisse
Amboise
37401
France
Buchar EvÏen
Inst. Experiment. Medicine
Acad. Sceinces Czech Rep.
VídeÀská 1083
Praha 4
142 20
Czech Republic
Bichet Andreas
Saarland University
Dept. of Biochemistry
Postfach 151150
Saarbrucken
66041
Germany
Burguiere Adeline
University of Birmingham
School of Biosciences
Edgbaston
Birmingham
B15 2TT
United Kingdom
Blobaum Anna
University of Michigan
Pharmacology
1150 W. Medical Center Drive,
MSRB III, Room 2200
Ann Arbor, MI
48109
Capdevila Jorge
Vanderbilt University
Nephrology
21st Avenue So.
Nashville, TN
37232-2372
S217
07 Index 11.6.2003 13:04 Stránka 218
Catania Stephania
CITSAL
Via Consolare Valeria, I
Messina
98125
Italy
Celier Chantal
INSERM U-473
84 rue General Leclerc
Le Kremlin Bicerno
94276
France
âervenková Katefiina
Medical Faculty of Palackého
University
Pathological Physiology
Hnûvotínská 3
Olomouc
77515
Czech Republic
Cesaro Claudia
Dr. Margarete Fischer
- Bosch Institute
Institute for Clinical Pharmacology
Auerbachstr. 112
Stuttgart
70376
Germany
Champion Paul
Northeastern University
Physics
110 Forsyth Street
Boston, MA
2115
Chang Chi K.
Hong Kong University
Sci. Technology
Dept. Chemistry
Clear Water Bay
Hong Kong
Chauret Nathalie
Merck Frosst Canada
Medicinal Chemistry
16711 TransCanada
Kirkland, QC
H9H 3LI
Canada
Chmela Zdenûk
Palacky University
Faculty of Medicine
Pathological Physiology
Hnûvotínská 3
Olomouc
775 15
Czech Republic
Chougnet Antoinette
University of Basel
Chemistry
St. Johanns-Ring 19
Basel
CH-4056
Switzerland
Costa Chiara
University of Messina
Faculty of Medicine
Via C. Valeria, 1
Messina
98126
Italy
Coecke Sandra
ECVAM
European Commission
Joint Research Centre
Via Fermi 1
Ispra
21020
Italy
Couture Manon
Laval University
Biochemistry
Marchand 4165
Quebec
G1K 7P4
Canada
Cohen Shimrit
Hebrew University
Organic Chemistry
Givat Ram Campus
Jerusalem
91904
Israel
da Silva Maria Eleonora Feracin
State University Campinas
Dept. Biochemistry
Cid.Universitária Zeferino Vaz s/n CP6109
Campinas
13083-970
Brazil
Collart Philippe
UCB S.A. Pharma Sector
Chemin du Foriest
Braine-l’Alleud
B-1420
Belgium
Coller Janet
University of Adelaide
Clinical and Experimental
Pharmacology
Level 5
Medical School North
Adelaide
5005
Australia
Coon Minor
Biological Chemistry
Ann Arbor, MI
48109-0606
Correia M. Almira
Universita of California
Cellular & Molecular Pharm.
513 Parnassus AJ
San Francisco
94143-0450
Cosme Jose
Astex Technology
436 Cambridge Science Park
Cambridge
CB40QA
United Kingdom
S218
Dallacosta Corrado
Universita’ di Pavia
Chimica Generale
Via Taramelli
Pavia
27100
Italy
Daniel Wladyslawa Anna
Polish Academy of Sciences
Institute of Pharmacology
Smetna 12
Krakow
31-343
Poland
Davydov Dmitri
University of Texas
Medical Branch
Pharmacology
and Toxycology
301 University Blvd.
Galveston
77555-1031
Dawidek-Pietryka Katarzyna
Medical Univercity
Toxicology
Chodzki 8
Lublin
20-093
Poland
Dawson John
University of South Carolina
Chemistry & Biochemistry
07 Index 11.6.2003 13:04 Stránka 219
631 Sumter Street
Columbia, SC
29208
de Graaf Chris
Pharmacochemistry
De Boelelaan 1083
Amsterdam
1081 HV
Netherlands
De Matteis Francesco
Birkbeck College
School of Biological and Chemical
Sciences
Malet Street
London
WCIE7HX
United Kingdom
de Visser Sam
Hebrew University
Organic Chemistry
Givat Ram Campus
Jerusalem
91904
Israel
Deprez Dominique
Aventis DMPK
13, quai Jules Guesde
BP 14 – 94403
Vitry sur Siene
France
Dlouhá Tereza
Charles University
Biochemistry
Hlavova 2030
Prague 2
128 40
Czech Republic
Dodhia Vikash
Biological Sciences
Exhibition Road
London
SW7 2AZ
United Kingdom
DoleÏal Tomá‰
3rd Faculty of Medicine
Pharmacology
Ruská
Prague 10
100 00
Czech Republic
DolÏan Vita
Faculty of Medicine
Vrazov trg 2
LJUBLJANA
SI-1000
Slovenia
Duisken Mike
Institute of Hygiene and Environmetal
Medicine, RWTH Aachen
Pauwelsstr. 30
Aachen
52074
Germany
Dunn Alexander
California Institute of Technology
Chemistry and Chemical
Engineering
MC 139-74
Pasadena, CA
91125
Fairhead Michael
Biological Sciences
Exhibition Road
London
SW7 2AZ
United Kingdom
Fantuzzi Andrea
Imperial College
Biological Sciences
Exhibition Road
London
SW7 2AZ
United Kingdom
Farghali Mohammad Hassan
Faculty of Medicine
Albertov 4
Praha 2
128 00
Czech Republic
Eagling Victoria
Quintiles Ltd.
DMPK + Bioanalysis
Research Av. Research Park
Edinburg
EH14 4 AP
United Kingdom
Fink Martina
Faculty of Medicine
Vrazov trg 2
Ljubljana
1000
Slovenia
Ellis Solo Wynne
University of Sheffield
Molecular Pharm. &
Pharmacogenetics
Sheffield
S10 2JF
United Kingdom
Flanagan Jack
Univeristy of Dundee
Biomedical Research Centre
Ninewells Hospital
Dundee
DD1 9SY
United Kingdom
Ericksen Spencer
West Virginia University
Basic Pharmaceutical Sciences
PO Box 9530
Morgantown
26505
Fon Tacer Klementina
Faculty of Medicine
Institute of bochemistry
Vrazov trg 2
Ljubljana
1000
Slovenia
Estabrook Ronald
UT Southwestern
Biochemistry
5323 Harry Hines Blvd
Dallas, Texas
75390
Franklin Michael
University of Utah
Pharmacology and
Toxicology
30 South 200 East, Rm 201
Salt Lake City, Utah
84112-5820
Fáberová Viera
VULM a.s.
Horná 36
Modra
900 01
Slovak Republic
Fuhr Uwe
Institute for Pharmacology,
University of Koeln
Clinical Pharmacology
Gleueler Strasse 24
Koeln
S219
07 Index 11.6.2003 13:04 Stránka 220
50931
Germany
Fujii-Kuriyama Yoshiaki
University of Tsukuba
TARA Centrum
1-1-1 Tennoudai
Tsukaba
305 8577
Japan
Fujino Hideki
Kowa Company Ltd.
Tokyo New Drug Research
Laboratories
2-17-43 Noguchicho
Higashimurayama
189-0022
Japan
Fulco Armand
UCLA Medical School
Biological Chemistry
P.O. Box 951737
Los Angeles
90095-1737
Funae Yoshihiko
Osaka City University Medical School
Chemical Biology
Asahimachi Abeno-ku Osaka
545-8585
Japan
Furge Laura
Kalamazoo College
Chemistry
1200 Academy St.
Kalamazoo
49006
Gal Joseph
University of Colorado
School of Medicine
4200 East 9th Avenue
Denver, CO
80262
Garrigues Alexia
Advanced Research L’Oréal
Metabolism Unit
1’Avenue Eugéne Schueller
Aulnay sous Bois
BP 22 - 93601
France
Georgescu Oana Ruxandra
SUB Bucharest
Allergology
Vlaicu Voda nr 13
Bl V63 Ap 32
Bucharest
7000
Romania
Gibson Gordon
University of Surrey
Biomedical Sciences
Stag Hill
Guildford
GU2 7XH
United Kingdom
Gilardi Gianfranco
Biological Sciences
South Kensington
London
SW7 2AY
United Kingdom
Gillam Elizabeth
University of Queensland
Physiology & Pharmacology
St. Lucia
Brisbane
4072
Australia
Gilbert Lawrence
University of N. Carolina
Dept. Biology
CB 3280 Chapel Hill, NC.
27599-3280
Goldfarb Peter
University of Surrey
School of Biomedical’s Life
Sciences
Stag Hill
Guilford, Surrey
GU2 7XH
United Kingdom
Gonzalez Frank. J.
National Cancer Institute
Building 37, Room 2A19
9000 Rockville Pike
Bethesda, MD
20892
Graham Sandra
U T Southwestern
Medical Center
Biochemistry
Dallas, TX.
75390-9038
S220
Gravel Maud
Centre de Recherche
en Cancérologie
Centre de Recherche
11 Cote du Palais
Québec
G1K 7P4
Canada
Groenhof Arend R.
Faculty of Sciences;
Division of Chemistry
De Boelelaan 1083
Amsterdam
1081 HV
Netherlands
Grubor Vladimir
University of Melbourne
Genetics
Royal Parade
Melbourne
3010
Australia
Guan Zhiwen
Himeji Institute of Technology
Dept. of Life Science
3-2-1 Kouto
Kamigori, Hyogo
679 1299
Japan
Guengerich F. Peter
Biochemistry
Nashville, TN
37232-0146
Guillouzo Andre
University of Rennes
INSERM U 456
2 ar Prof. L. Bernard
Rennes
35043
France
Hada‰ová Eva
Pharmacological Institute
Jo‰tová 10
Brno
662 43
Czech Republic
Haduch Anna
Dept. of Pharmacokinetics
Smetna 12
Krakow
07 Index 11.6.2003 13:04 Stránka 221
31-343
Poland
21227
Hainzl Dominik
F. Hoffmann - La Roche
Grenzacherstr. 124
PRBD-E Bld. 68/336
4070 Basel
Switzerland
Hodek Petr
Charles University
Biochemistry
Hlavova 2030
Prague 2
128 40
Czech Republic
Hakkola Jukka
University of Oulu
and Toxicology
POB 5000 (Aapistie 5)
Oulu
90014
Finland
Halpert James
UTMB
Pharm. Tox.
Galveston
77555-1031
Hodis Jifií
Charles University
Pharmacy
Heyrovského 1203
Hradec Králové
500 12
Czech Republic
Hudeãek Jifií
Charles University
Biochemistry
Albertov 2030
Praha 2
128 40
Czech Republic
Helia Otto
Comenius University
Fac. Pharmacy
Mol. Biol. Drugs
Kalinãakova 8
Bratislava
832 32
Slovak Republic
Hughes Kelvin
Amersham Biosciences
Drug Discovery
Forest Farm
Cardiff
CF14 7EG
United Kingdom
Helvig Christian
Cytochroma Inc.
330 Cochrane Drive
Markham, ON
L3R 8E4
Canada
Hui Bon Hoa Gaston
INSERM U-473
84 rue General Leclerc
Le Kremlin Bicerno
94276
France
Henderson Colin
Cancer Research Centre
Biochemical Research Centre
Ninewells Hospital
Dundee
DDI 9SY
United Kingdom
Hyland Ruth
Pfizer Global Research
& Development
P.D.M.
Ramsgate Road
Sandwich, Kent
CT13 9NJ
United Kingdom
Herman Darja
Faculty of Medicine
Vrazov trg 2
Ljubljana
SI-1000
Slovenia
Hewitt Nicky
In Vitro Technologies
1450 S. Rolling Rd.
Baltimore
Idle Jeff
University of Bern, Switzerland
U Haje 1651
Roztoky u Prahy
25263
Czech Republic
Ingelman-Sundberg Magnus
Karolinska Institutet
S221
Molecular Toxicology
Nobels v 13
Stockholm
171 77
Sweden
Ishimura Yuzuru
The University of Texas
Health Science Center
at San Antonio
Biochemistry
7703 Floyd Curl Drive
San Antonio, Texas
78229-3900
Ivanov Alexis
Lab. Mol. Graph. & Drug Design
Pogodinskaya str. 10
Moscow
119121
Russia
Iwasaki Masaru
Daiichi Pharmaceutical Co., Ltd.
Drug Metabolism Physicochemical
Property Research Laboratory
16-13, Kita-Kasai 1-Chome
Edogawaku, Tokyo
134-8630
Japan
Iyanagi Takashi
Himeji Institute
of Technology
3-2-1 Kouto
Kamigori, Hyogo
680 1299
Japan
Jáchymová Marie
Laboratory of Molecular
Cardiology
U Nemocnice 2
Praha 2
12808
Czech Republic
Jaremko Ma∏gorzata
Institute of Clinical
Pharmacology
Auerbachstr.108
Stuttgart
Germany
Jetter Alexander
University of Koeln
Gieveler str. 24
Koeln
07 Index 11.6.2003 13:04 Stránka 222
D-50931
Germany
Jgoun Alexander
Centre of Bioengineing biology
Barvihinskaya, 8-2-12
Moscow
121596
Russia
Johnson Eric F.
The Scripps Research Institute
MEM 255
10550 N. Torrey Pines Rd
La Jolla
92037
Canada
Jourdan Nathalie
Pfizer
Research Technology
3-9 Rue de la Loge
Fresnes
94265
France
Jung Christiane
Max-Delbrück-Center
for Molecular Medicine
Protein Dynamics
Robert-Rössle-Strasse 10
Berlin
13125
Germany
Juvonen Risto
University of Kuopio
Pharmacology
and Toxicology
Harjulantie 1
Kuopio
70211
Finland
Kamataki Tetsuya
Hokkaido University
Pharmaceutical Sciences
Kita 12, Nishi - 6, Kita-ku
Sapporo
060-0812
Japan
Kamp Sarah
InPharmatica Target Evaluation
2 Royal College
NW1 0TV
London
United Kingdom
Karabec Martin
Charles University
Biochemistry
Hlavova 2030
Prague 2
128 40
Czech Republic
Karlgren Maria
Molecular Toxicology, IMM
Nobels väg 13
Stockholm
171 77
Sweden
Keizers Peter
Vrije Universiteit
Pharmacochemistry,
Molecular Toxicology
De Boelelaan 1083
Amsterdam
1081 HV
Netherlands
Kelly Steven
University of Wales
Aberystwyth
Wolfson Laboratory
of P450 Biodiversity
Penglais Hill
Aberystwyth
SY23 3DA
United Kingdom
Kent Ute
Pharmacology
1150 West Medical Ctr. Dr
Ann Arbor, Michigan
48109
Kenyon George
College of Pharmacy
428 Church St.
Ann Arbor, MI
48109-1056
Kikuchi Hideaki
IDAC Tohoku University
Molecular Genetics
4-1 Seiryo-machi
Sendai
980-8575
Japan
Kikuta Yasushi
Fukuyama University
Applied Biological Science
Gakuencho-1
Fukuyama
729-0292
Japan
S222
Kim Kyoung-Ah
Kyung Hee University
East-West Medical Research Institute
Hoegidong
Seoul
130-701
South Korea
Kimura Shigenobu
3-2-1 Kouto
Kamigori, Hyogo
678-1297
Japan
Klein Kathrin
Dr. Margarete Fischer-Bosch
Institute for Clinical
Pharmacology
Auerbachstr. 112
Stuttgart
D-70376
Germany
Koblas Tomá‰
Charles University
Biochemistry
Hlavova 2030
Prague 2
128 40
Czech Republic
Kaloshyna Natalia
Institute of Biochemestry
BLK 50
Grodno
230017
Belarus
Kominami Shiro
Hiroshima University
Integrated Arts and Sciences
1-7-1 Kagamiyama
Higashihiroshima
739-8521
Japan
Kondrová Eli‰ka
Natl. Inst. Public Health
Ctr. Industrial Hygiene
and Occupational Health
·robárova 48
Praha10
100 42
Czech Republic
Koskinen Mikko
Orion Pharma
Depr. of Non-Clinical
Pharmacokinetics
P.O.Box 65
Espoo
07 Index 11.6.2003 13:04 Stránka 223
FIN 02101
Finland
Kousalova Lucie
Palacky University, Faculty
of Medicine
Dpt. of Pharmacology
Hnevotinska 3
Olomouc
77515
Czech Republic
Kunze Kent
Medicinal Chemistry
Box 357610
Seattle, WA
98195
Kutinová Canová Nikolina
Charles University, Medicine
Albertov 4
Praha 2
120 00
Czech Republic
Kuznetsov Vadim
Laboratory of Biosensor Analysis
Pogodinskaya, 10
Moscow
119121
Russia
Lah Ljerka
National Institute of Chemistry
Biochemistry
Hajdrihova 19
Ljubljana
SI-1000
Slovenia
Lachaud Antoine Amaury
Centre de Recherche
11 Cote du Palais
Québec
G1K 7P4
Canada
Laizier Marianne
Biologie
rue Mac Mahon
Québec
G1R 2J6
Canada
Lam Y. W. Francis
Univ. TX Health Science Center
at San Antonio
Pharmacology (MSC 6220)
San Antonio, TX
78229-3900
Lamb David C.
University of Wales Aberystwyth
Institut of Biological Sciences
Penglais Hill
Aberystwyth
SY23 3DAS
United Kingdom
Lampe Jed
Dept. of Medicinal Chemistry
Box 357610
Seattle
98195
Lange Reinhard
INSERM
U128
1919, route de Mende
Montpellier
F-34293
France
LaÀková Martina
Charles University
Biochemistry
Hlavova 2030
Prague 2
128 40
Czech Republic
Lazar Andreas
University of Cologne
Institute for Pharmacology
Gleueler Str. 24
Cologne
50931
Germany
Lee-Robichaud Peter
Chemistry
Dainton Bldg, Brookhill
Sheffield
S3 7HF
United Kingdom
Lisitsa Andrey
Institute of Biomedical
Chemistry
Lab. Mol. Graph. & Drug Design
Pogodinskaya str. 10
Moscow
S223
119121
Russia
Loeper Jacqueline
INSERM
U 538
27 rue de Chaligny
Paris
75012
France
Lupp Amelie
University of Jena
Inst. of Pharmacology
and Toxicology
Nonnenplan 4
Jena
D-07743
Germany
Makris Thomas
University of Illinois
Biochemistry
505 S Goodwin
Urbana
61801
Malmebo Sarah
Molecular Toxicology, IMM
Nobels vag 13
Stockholm
17177
Sweden
Mancy Annah
Pfizer
Pharmacokinetics,
Dynamics and Metabolism
3-9 rue de la Loge,
BP100
Fresnes
94265
France
Mansuy Daniel
Universite Paris 5
UMR 8601
45 Rue des Saints-Pieres
Paris, CEDEX 06
75270
France
Marill Julie
INSERM U-496
Centre Hayem,
Hôpital St Louis
1 avenue Claude Vellefaux
Paris
75010
France
07 Index 11.6.2003 13:04 Stránka 224
Marková Vladimíra
Charles University
Biochemistry
Hlavova 2030
Prague 2
128 40
Czech Republic
Marshall Ker
University of Leicester
Biochemistry
University Road
Leicester
LE1 7RH
United Kingdom
Marshall Toby
Quintiles Ltd.
DMPK + Bioanalysis
Edinburg
EH14 4 AP
United Kingdom
Martásek Pavel
Charles University
General Faculty Hospital
Lab. Inherited Metabol. Dis.
Ke Karlovu 2
Praha 2
Czech Republic
Martínek Václav
Charles University
Biochemistry
Hlavova 2030
Prague 2
128 40
Czech Republic
Martínková Jifiina
Charles University
Medical Faculty
·imkova 670
Hradec Králové
500 02
Czech Republic
Martinez Vanesa
National Institute for Cellular
Biotechnology
National Cell and Tissue Culture
Centre
R&D Building, DCU Campus
Glasnevin
Dublin 9
Ireland
Marvasi Luigi
University of Bologna
Sanità Pubblica Veterinaria
Via Tolara di Sopra 50
Ozzano Emilia (Bologna)
40064
Italy
Am Feld 32
Grafing
D-85567
Germany
Masters Bettie Sue
The University of Texas Health
Science Center at San Antonio
Biochemistry
San Antonio, TX
78229-3900
Micuda Stanislav
Faculty of Medicine
Pharmacology
Simkova 870
Hradec Kralove
50038
Czech Republic
Matsui Kazuhiro
Tsuruga Institute of
Biotechnology/TOYOBO
34-3-1 103
Tsuruga-shi
914-0038
Japan
Mik‰anová Markéta
Charles University
Biochemistry
Hlavova 2030
Prague 2
128 40
Czech Republic
Maurel Patrick
INSERM U 128
1919 Route de Mende
Montpellier
34293
France
Miko Milan
Slovak Technical University
Faculty of Chemical Technology
Dept. of Biochemistry and
Microbiology
Radlinského 9
Bratislava
812 37
Slovak Republic
McLaughlin Lesley
Univeristy of Dundee
Biomedical Research Centre
Ninewells Hospital
Dundee
DD1 9SY
United Kingdom
McLean Kirsty
Biochemistry
University Road
Leicester
LE1 7RH
United Kingdom
Meilleur Flora
EMBL
Neutron Diffraction
6 rue Jules Horowitz
Grenoble
38042
France
Meinhold Peter
California Institute of Technology
Chemistry and Chemical Engineering
1200 E, California Blvd.
Pasadena, CA
91125
Meyring Michael
Merck KGaA
Drug Metabolism
S224
Mitani Fumiko
Keio University School Medicine
Biochemistry
35 Shinanomach
Shinjuku-ku, Tokyo
160-8582
Japan
MonostoryKatalin
Chemical Research Center
Biochemical Pharmacology
Pusztaszeri 59-68
Budapest
H-1026
Hungary
Moreau Magali
Laboratoire de Chimie et Biochimie
Pharmacologiques et Toxicologiques
45 rue des Saint Peres
Paris cedex 06
75270
France
Morishima Isao
Kyoto University
Department of Molecular
Engineering
Yoshida-Honnmachi, Sakyo-ku
Kyoto City
606-8501
Japan
07 Index 11.6.2003 13:04 Stránka 225
Munro Andrew
Biochemistry
Adrian Building, University Road
Leicester
LE1 7RH
United Kingdom
Mürdter Thomas E.
-Institute of Clinical
Pharmacology
Auerbachstr. 112
Stuttgart
D-70376
Germany
Nakajima Miki
Kanazawa Univeristy
Faculty of Pharmaceutical
Sciences
Takara-machi 13-2
Kanazawa
920-0934
Japan
S3 7HF
United Kingdom
Oesch Franz
University of Mainz
Institute of Toxicology
Obere Zahlbacher Str. 67
Mainz
D-55131 Mainz
Germany
Ohkawa Hideo
Kobe University
Environmental Genomics
Rokkodaicho 1-1
Nada, Kobe
657-8501
Japan
Omura Tsuneo
Kyushu University
7-17-7 Hinosato
Munakata, Fukuoka
811 34 25
Japan
Trikalon 224
Karditsa
43100
Greece
Parker Josie
University of Wales
Inst. of Biological Sciences
Penglais
Abersystwyth
SY23 3DA
United Kingdom
Parkinson Andrew
XenoTech, LLC
16825 West 116th Street
Lenexa, KS
66219
Parton Andrew
Celltech R&D Ltd.
216 Bath Road
Slough
SL1 4EN
United Kingdom
Narasimhulu Shakunthala
University of Pennsylvania
HDSR/Richards Bldg.
36th & Hamilton Walk
Philadelphia, PA
19104
Ortiz de Montellano Paul
University of California,
San Francisco
Pharmaceutical Chemistry
60016th Street
San Francisco
94143-2280
Canada
Pascussi Jean Marc
INSERM
UMR128
1919 route de Mende
Montpellier
34293
France
Ni Jinsong
Allergan
PKDM
2525 Dupont Dr.
Irvine/CA
92612
Ozcubukow Salih
RWTH Aachen
Inst. Organic Chem.
Prof. Pirlet Str. 1
Aachen
52074
Germany
Pastera Jifií
Inst. Exper. Biopharmacy
Academy of Sciences, Czech Rep.
Heyrovského 1207
Hradec Králové
500 02
Czech Republic
Nobilis Milan
Heyrovského 1207
Hradec Králové
500 02
Czech Republic
Ozdamar Elcin Deniz
Ankara University Faculty of Pharmacy
Toxicology
Dö Gol
Ankara
6100
Turkey
Paxton James
The University of Auckland
& Clinical Pharmacology
Auckland
92019
New Zealand
Novakovic Jasmina
Hrdliãkova 2188
Praha 4
140 00
Czech Republic
O’Donnell Emily
Chemistry
Sheffield
Pandova Bozhidarka
Institute of Physiology, BAS
Drug Toxicology
Acad.G.Bonchev St., bl.23
Sofia
1113
Bulgaria
Pappas Ioannis
Univeristy of Thessaly
Veterinary Medicine
S225
Pearson Josh
Medicinal Chemistry
Box 357610
Seattle, WA
98195
Pelkonen Olavi
University of Oulu
Dept. of Pharmacology and
Toxicology
07 Index 11.6.2003 13:04 Stránka 226
POB5000
Oulu
90014
Finland
Peterson Julian
UT Southwestern Medical Center
Biochemistry
Dallas, TX
75390-9038
Petrie Derek
Quintiles Ltd.
DMPK + Bioanalysis
Edinburg
EH14 4 AP
United Kingdom
Pfaar Ulrike
Novartis Pharma AG
Preclinical Safety / ADME
Basel
CH-4002
Switzerland
Pikuleva Irina
University of Texas Medical
Branch
Galveston, TX
77555-1031
Podust Larissa
Biochemistry
23rh South and Pierce
Nashville, TN
37232-0146
Pokrajac Milena
University of Belgrade
Faculty of Pharmacy
POB. 146
Vojvode Stepe 450
Belgrade 1100
Serbia and Montenegro
Poljaková Jitka
Charles University
Biochemistry
Hlavova 2030
Prague 2
128 40
Czech Republic
Pons Nicolleta
of Anatomy, Pharmacology
and Forensic Medicine
Via Pietro Giuria 13
Torino
10129
Italy
Poulos Thomas
University of California, Irvine
Molecular Biology and Biochemistry
2206 Natural Sciences 1
Irvine, CA
02697-3900
Price Julie
In Vitro Technologies
1450 S. Rolling Rd.
Baltimore, MD
21227
Provazníková Dana
Charles University
Biochemistry
Hlavova 2030
Prague 2
128 40
Czech Republic
Rahnasto Minna
Univ. of Kuopio
Pharm. Toxicol.
Harjulantie 1A
Kuopio
70210
Finland
Raman C. S.
University of Texas
Medical School
Dept. of Biochemistry and Molecular
Biology
Houston, TX
Rautio Arja
University of Oulu
Dept. Pharmacology and
Toxicology
P.O.Box 5000
Oulu
FIN-90014
Finland
ReÏen Tadeja
Faculty of Medicine
Vrazov trg 3
Ljubljana
1000
Slovenia
S226
Riccardi Benedetta
Chiesi Farmaceutici S. p. A.
Pharmacokinetics
Via Palermo 26/A
Parma
43100
Italy
Ridderström Marianne
AstraZeneca R&D Mölndal
DMPK & Bioanalytical
Chemistry
Pepparedsleden
Mölndal
43183
Sweden
Riedel Jens
Aventis Pharma
DMPK
Industriepark Höchst
Frankfurt
65926
Germany
Rieger Michael
Medical Faculty, Technical
University Dresden
Institute of Immunology
Fetscherstrasse 74
Dresden
1307
Germany
Richter Tanja
Dr. Margarete Fischer - Bosch
Institute
Institute for Clinical Pharmacology
Auerbaehstr. 112
Stuttgart
70376
Germany
Richter-Addo George
University of Oklahoma
Chemistry and Biochemistry
620 Parrington Oval
Norman, Oklahoma
73019
Rodriguez-Antona Cristina
Karolinska Institute
Environmental Medicine
Mol. Toxicology
Nobels väg 13
Stockholm
17177
Sweden
Roitel Olivier
Biochemistry
07 Index 11.6.2003 13:04 Stránka 227
University Street
Leicester
LE1 6RR
United Kingdom
Roman Linda
The University of Texas Health
Science Center at San Antonio
Biochemistry
San Antonio, TX
78229-3901
Ronis Martin
University of Arkansas for Medical
Sciences
1120 Marshall St./R2052
Little Rock, Arkansas
72202
Roymans Dirk
Johnson & Johnson
Preclinical Pharmacokinetics
Turnhoutseweg 31
Beerse
2340
Belgium
Royo González Inmaculada
Merck Sharp & Dohme
CIBE
Josefa Valcarcel 38
Madrid
28027
Spain
Rozman Damjana
University of Ljubljana
Med.Center for Mol. Biology
Vraza trg 2
Ljubljana
1000
Slovenia
Ruckpaul Klaus
Max-Delbrueck-Center of Molecular
Medicine
Robert Roessle Str. 10
Berlin
13122
Germany
Rydberg Patrik
Lund University
Theoretical Chemistry
P.O. Box 124
Lund
S-221 00
Sweden
Rynn Caroline
Merck Sharp & Dohme
Medicinal Chemistry
Eastwik Road
Harlow
CM20 2QK
United Kingdom
Sagami Ikuko
Tohoku Univeristy
Inst. of Multidisciplinary Research
Aoba-ku Katahira 2-1-1Sendai
980-8577
Japan
Samuel Dateh
Sustainable Integrated Develompent
Foundation
Human Right Section
Cantonments
Accra
233
Ghana
Sanchez Rubiales Manuel
Merck Sharp & Dohme
CIBE
Josefa Valcarcel 38
Madrid
28027
Spain
Santolini Jerome
CEA
DBJC-SBE
CEA-Saclay
Gif Sur Yvette
91191 cedex
France
Sassone Carlo
Imperial College
Biological Sciences
4 Regency Street
London
SW1P 4BZ
United Kingdom
Scott Emily
University of Texas
Medical Branch
Galveston, TX
77555-1031
Sen Alaattin
Pamukkale University
Biology
Kinikli Campus
S227
Denizli
20017
Turkey
Sevrioukova Irina
University of California, Irvine
Molecular Biology and
Biochemistry
3205 Bio Sci II
Irvine, CA
92697
Shaik Sason
Hebrew University
Department of Organic Chemistry
Givat Ram Campus
Jerusalem
91904
Israel
Sharma Pankaz
Hebrew University
Department of Organic Chemistry
Givat Ram Campus
Jerusalem
91904
Israel
Sheikh Qaiser
Chemistry
Sheffield
S3 7HF
United Kingdom
Shimizu Toru
Tohoku Univeristy
Inst. of Multidisciplinary Research
Aoba-ku Katahira 2-1-1
Sendai
980-8578
Japan
Shimoji Miyuki
Inst. of Environmental Medicine
Division of Biochemical
Toxicology
Stockholm
S-17177
Sweden
Shumyantseva Victoria
Pogodinskaya, 10
Moscow
119121
Russia
Schilling Boris
Givaudan
07 Index 11.6.2003 13:04 Stránka 228
Bioscience
Ueberlandstrasse 138
Duebendorf
8600
Switzerland
Schlichting Ilme
Max Planck Institute for Medical
Research
Department of Biomolecular
Mechanisms
Jahnstr. 29
Heidelberg
69120
Germany
Schuler Mary
Cell and Structural Biology
1201 W. Gregory Dr., 161 ERML
Urbana, IL
61801
Schwaneberg Ulrich
International University Bremen
Campus Ring 1
Bremen
28759
Germany
Sidorova Yulia Alexandrovna
Institute of Molecular Biology and
Biophysics, Siberian Division
of Russian Academy of Medical
Sciences
Laboratory of Biochemistry
of Foreign Compounds
Timakova str., 2
Novosibirsk
630117
Russia
Sielaff Bernhard
Martin-Lunther University Halle
Inst. of Microbiology
Kurt-Mothes Str. 3
Halle
06120
Germany
Silvari Virginia
Univeristy of Messina
Faculty of Pharmacy
SS. Annunziata
Messina
98127
Italy
·imánek Vilím
Palacky University
Faculty of Medicine
Institutek of Medical Chemistry
Hnûvotínská 3
Olomouc
775 15
Czech Republic
Skálová Lenka
Hradec Králové
Pharmacology
Heyrovského 1204
Hradec Králové
501 05
Czech Republic
Slaviero Kellie
University of Sydney
Pharmacology
Bosch Building
Sydney
2006
Australia
Sligar Stephen
Biochemistry
505 S. Goodwin Street
Urbana
IL 61801
Slíva Jifií
Charles University
Medical Faculty
Pharmacology
Ruská 87
Praha 10
100 00
Czech Republic
Souãek Pavel
·robárova 48
Praha10
100 42
Czech Republic
Stark Katarina
Unive. Uppsala
Div. Biochem. Pharmacol.
Box 591
Uppsala
751 24
Sweden
Stiborová Marie
Charles University
Biochemistry
Hlavova 2030
Prague 2
128 40
Czech Republic
S228
Stiborová-Rupertová Martina
Charles University
Biochemistry
Hlavova 2030
Prague 2
128 40
Czech Republic
·tûrba Martin
Charles University
Fac. Medicine HK
Institut Pharmacology
·imkova 870
Hradec Králové
500 38
Czech Republic
Straub Wesley
UCSF
Chem., Chem. Biol.
1357A Page
San Francisco, CA
94117
Strobel Henry W.
UT Medical School
Biochemistry and Molecular
Biology
6431 Fannin
Houston, TX
77030
Stfie‰tíková Petra
Charles University, Medicine
Albertov 4
Praha 2
120 00
Czech Republic
Sukhodub Andrey
University of Portsmouth
Biophysics Laboratories
White Swan Road
Portsmouth
PO1 2 DT
United Kingdom
Sutcliffe Michael
Biochemistry
University Road
Leicester
LE1 7RH
United Kingdom
Svoboda Zbynûk
Heyrovského 1207
Hradec Králové
07 Index 11.6.2003 13:04 Stránka 229
500 02
Czech Republic
Swart Pieter
University of Stellenbosch
Biochemistry
van der Byl
Stellenbosch
7600
South Africa
Swart Amanda
University of Stellenbosch
Biochemistry
van der Byl
Stellenbosch
7600
South Africa
Szklarz Grazyna
West Virginia University
Basic Pharmaceutical Sciences
Health Sciences Center,
P.O. Box 9530
Morgantown, VW
26506
Szotáková Barbora
Hradec Králové
Pharmacology
Heyrovského 1205
Hradec Králové
502 05
Czech Republic
Takyi Stephen
Sustainable Integrated Develompent
Foundation
Human Right Section
Cantonments
Accra
233
Ghana
Tarès Sophie
INRA
UMR n°1112 -API lab.123 Bd. F. Meilland, BP2078
Antibes cedex
6606
France
Tejura Bina
Marix Drug Development
Rhodfa Marics, Ynysmaerdy
Llantrisant,
S Wales
CF72 8UX
United Kingdom
Tesafiová Martina
Hospital Kladno
Paediatric Dept.
Vanãurova 1548
Kladno
272 59
Czech Republic
Theisen Michael
Abbott Laboratories
Structural Biology
100 Abbott Park Road
Abbott Park, IL
60064-6098
Thompson John
University of Colorado
Pharmaceutical Sciences
4200 E. 9th Ave, Box C238
Denver, CO
80262
Tomková SoÀa
Charles University
Biochemistry
Hlavova 2030
Prague 2
128 40
Czech Republic
Tosha Takehiko
Kyoto University
Molecular Engineering
Yoshida Honmachi
Kyoto city
606-8501
Japan
Trejtnar Franti‰ek
Charles University
Pharmacy
Heyrovského 1203
Hradec Králové
500 12
Czech Republic
Tschirret-Guth Richard
Merck & Co.
Drug Metabolism
PO Box 2000
Rahway, NJ
07065
Tsuchiya Yuki
Kanazawa Univeristy
Faculty of Pharmaceutical
Sciences
Takara-machi 13-1
Kanazawa
S229
9 200 934
Japan
Turpeinen Miia
University of Oulu
Dept. of Pharmacology
and Toxicology
P.O.BOX
Oulu
90014
Finland
Tyndale Rachel
University of Toronto
Pharmacology
1 King Collage
Toronto
M55 1A8
Canada
Ueng Yune-Fang
National Research Inst. of Chinese
Medicine
155-1, Li-Nong St. Sec 2
Taipei
112
Taiwan
Urban Philippe
Centre de Genetique Moleculaire
CNRS
avenue de la Terrasse
Gif-sur-Yvette
91190
France
Urlacher Vlada
Insitute of Technical Biochemistry
University of Stuttgart
Allmandring 31
Stuttgart
70569
Germany
Václavíková Radka
·robárova 48
Praha10
100 42
Czech Republic
Vazquez-Duhalt Rafael
National University
of Mexico
Biotechnology Institute
Av. Universidad 2001
Cuernavaca, Morelos
62250
Mexico
07 Index 11.6.2003 13:04 Stránka 230
Velík Jakub
Charles University
Pharmacy
Heyrovského 1203
Hradec Králové
500 12
Czech Republic
Department of General
Toxicology
Ezhena Pot’e 14
Kyiv
3057
Ukraine
Vereczkey László
Chemical Research Center
Biochemical Pharmacology
Pusztaszeri 59-67
Budapest
H-1025
Hungary
Wade Rebecca
European Media Laboratory
Molecular and Cellular
Modeling
Schloss-Wolfs Brunnenweg 33
Heidelberg
69118
Germany
Vermeir Marc
Johnson & Johnson
Preclinical Pharmacokinetics
Turnhoutseweg 30
Beerse
2340
Belgium
Wang Regina
Merck Research Laboratories
Drug Metabolism
PO Box 2000
Rahway, NJ
7065
Vignati Luisella
Pharmacia Italia S.p.A.
Global Drug Metabolism
Viale Pasteur, 10
Nerviano
20014
Italy
Ward Richard
Biological NMR Centre
University Road
Leicester
LE1 9HN
United Kingdom
Viitala Pirkko
University of Oulu
and Toxicology
P.O.Box 5000
(Aapistie 5)
Oulu
FIN-90014
Finland
Waskell Lucy
Aneasthesiology
2215 Fuller Rd. 11 R
Ann Arbor, MI
48105
von Weymarn Linda
Pharmacology
1150 W. Medical Center Dr. Room
2200 MSRB III
Ann Arbor, MI
48109
Vondrá‰ková Lenka
Charles University
Biochemistry
Hlavova 2030
Prague 2
128 40
Czech Republic
Voronina Alla
& Toxicology
Watanabe Minro
Iwate Pref. Univ.
Social Welfare
Takizawa Shi
Iwate Ken
020-0193
Japan
Waterman Michael
Vanderbilt
Univ. Sch. Med
Biochemistry
607 Light Hall
Nashville, TN
37232-0146
Wauthier Valérie
UCL
PMNT
Avenue Mounier
Bruxelles
S230
1200
Belgium
Waxman David J.
Boston University
Dept. Of Biology
Boston, MA
Webber Guy
Biodynamics Research Ltd.
Drug Metabolism and
Pharmacokinetics
Pegasusway
Rusmoen
NNIO GER
United Kingdom
Wensing Georg
Bayer AG
BHC-PH-PD CPD
Aprather Weg 18a
Wuppertal
D-42096
Germany
Westlake Andrew
Biological NMR Centre
University Road
Leicester
LE1 9HN
United Kingdom
Woggon Wolf-D.
University of Basel
Chemistry
St. Johanns-Ring 19
Basel
CH-4056
Switzerland
Wojcikowski Jacek
Polish Academy of Sciences
Dept. of Pharmacokinetics
Smetna 12
Krakow
31-343
Poland
Wong Tuck Seng
International University Bremen
Campus Ring 1
Bremen
28759
Germany
Wong Luet
University of Oxford
Department of Chemistry, Inorganic
Chemistry Laboratory
South Parks Road
Oxford
07 Index 11.6.2003 13:04 Stránka 231
OX1 3QR
United Kingdom
Yadav Jagjit
University of Cincinnati College
of Medicine
Environmental Health,
Molecular Toxicology Division
3223 Eden Ave
Cincinnati
45267-0056
Yamamoto Keita
3-2-1 Kouto
Kamigori, Hyogo
678-1299
Japan
Yanev Stanislav
Institute of Physiology, BAS
Drug Toxicology
Acad.G.Bonchev St., bl.23
Sofia
1113
Bulgaria
Yin Hequn
Novartis Pharma AG
Preclinical Safety
One Health Plaza
East Hanover
07936
Auerbachstr. 112
Stuttgart
70376
Germany
Yokoi Tsuyoshi
Kanazawa Univeristy
Faculty of Pharmaceutical Sciences
Takara-machi 13-3
Kanazawa
920-0934
Japan
Zídek Zdenûk
Inst. Experiment. Medicine
Acad. Sciences Czech Rep.
VídeÀská 1083
Praha 4
142 20
Czech Republic
Yost Garold
University of Utah
30 South 200 East, Rm 201
Salt Lake City, Utah
84112-5820
Zima Tomá‰
Charles University
Med. Faculty I
Inst. Clin. Biochem. & Labor.
Diagnostics
U Nemocnice 2
Praha 2
12000
Czech Republic
Yun Chul-Ho
Pai-Chai University
Genetic Engineering
439-6 Doma-dong, Seo-ku
Taejon
302-735
South Korea
Zanger Ulrich M.
Dr. Margarete Fischer-Bosch Institute
of Clinical Pharmacology
S231
Zukunft Jörg
Institute of Clinical
Pharmacology
Auerbachstr. 112
Stuttgart
D-70376
Germany
07 Index 11.6.2003 13:04 Stránka 232
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Together, they shorten the gap
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